Chimeric protein comprising non-toxic pseudomonas exotoxin a and type iv pilin sequences

ABSTRACT

The invention provides chimeric proteins comprising a non-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loop sequence, wherein the Type IV loop sequence is inserted within the non-toxic Pseudomonas exotoxin A. The invention also provides polynucleotides encoding the chimeric proteins, and compositions comprising the polynucleotides or the chimeric proteins. The invention also provides methods for using the chimeric proteins, polynucleotides and compositions of the invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional ApplicationNo. 60/257,877 filed Dec. 21, 2000, the contents of which isincorporated herein by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Not Applicable.

BACKGROUND OF THE INVENTION

[0003] Type IV pilin is the major subunit of the pilus or pili which arefilamentous structures covering many microorganisms including bacteriaand yeast. Among these microorganisms, many pathogenic species expressType IV pilins, including, e.g., P. aeruginosa, N. meningitides, N.gonorrhoeae, Vibro cholera, and Pasteurella multocidam. The first stepin infection with these pathogenic microorganisms is adherence to targetcells through the pili. In particular, Type IV pilins of Pseudomonasaeruginosa bind to asialoGM1 receptors on epithelial cells (Saiman etal., J. Clin. Invest. 92(4):1875-80 (1993); Sheth et al., 11(4):715-23(1994); Imundo et al., Proc. Natl. Acad. Sci. USA, 92(7):3019-23 (1995);Hahn, Gene 192(1):99-108 (1997)). Thus, the pili of these microorganismsare a major virulence factor, and result in colonization by pathogenicmicroorganisms and infections in humans.

[0004] For example, Pseudomonas aeruginosa causes between 10% and 20%infections in most hospitals. Pseudomonas infection is common amongpatients with cystic fibrosis, burn wounds, organ transplants, andintravenous-drug addiction. Pseudomonas infections can lead to seriousconditions, such as endophthalmitis, endocarditis, meningitis,pneumonia, and septicemia. In particular, colonization of cysticfibrosis (CF) individuals with Pseudomonas aeruginosa represents asignificant negative milestone in the progression of this disease. Oncecolonized, patients are subject to the damaging effects of varioussecreted virulence factors and to the inflammatory response of the hostimmune system.

[0005] Type IV pili are composed of pilin polymers arranged in a helicalstructure with five subunits per turn (Forest et al., Gene 192(1): 165-9(1997); Parge, Nature 378(6552):32-8 (1995)). The portion of the pilinprotein responsible for cell binding is found near the C-terminus (aminoacids 122-148) in a β-turn loop subtended from a disulfide bond(Campbell et al., Biochemistry 36(42):12791-801 (1997); Campbell et al.,J. Mol. Biol. 267(2):382-402 (1997); Hazes et al., J. Mol. Biol.299(4):1005-1017 (2000); McInnes et al., Biochemistry 32(49):13432-40(1993)). For P. aeruginosa, a 12 or 17 amino acid sequence (depending onthe strain) in this loop interacts with receptors on epithelial cells.For CF individuals, the overproduction of the R domain of mutant cysticfibrosis transmembrane conductance regulator (CFTR) can lead to anincreased level of asialoGM1 and, accordingly, an increased binding ofP. aeruginosa (Imundo et al., Proc. Natl. Acad. Sci. USA 92(7):3019-23(1995); Saiman et al., J. Clin. Invest. 92(4):1875-80 (1993); Bryan etal., Am. J. Respir. Cell Mol. Biol. 19(2):269-77 (1998); Imundo et al.,Proc. Natl. Acad. Sci. USA 92(7):3019-23 (1995); Saiman et al., J. Clin.Invest. 92(4):1875-80 (1993)). Functional studies of pilin haveindicated that only the last pilin subunit (the tip) of a pilusinteracts with epithelial cell receptors (Lee et al., Mol. Microbiol.11(4):705-13 (1994)).

[0006] To date, efforts to produce an effective anti-pilin vaccine havenot been very successful. In part, this limited success is because themost immunogenic portion of the protein (the middle) does not generateantibodies that interfere with adhesion. Unfortunately, the C-terminalloop of pilin is not very immunogenic, and high titer responses haveonly been reported with the use of strategies that employ multipledisplay copies of the loop sequence (Hahn et al., Behring. Inst. Mitt.(98):315-25 (1997)). For CF patients, strategies to inhibit Pseudomonascolonization are considered an important element in reducing themorbidity normally associated with the development of chronic infections(Tang et al., Infect. Immun. 63(4):1278-85 (1995); Li et al., Proc.Natl. Acad. Sci. USA 94(3):967-72 (1997); Tang et al., Infect. Immun.63(4):1278-85 (1995) Doig, P. et al., Infect Immun 58(1): 124-30 (1990);El-Zaim, H. S. et al. Infect Immun 66(11):5551-4 (1998)).

[0007] Accordingly, there is a need to develop compositions for reducingor preventing infections by pathogenic microorganisms including, inparticular, Pseudomonas aeruginosa. Embodiments of this inventionaddress this and other needs.

SUMMARY OF THE INVENTION

[0008] Embodiments of the invention provide chimeric proteins comprisinga non-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, wherein the Type IV pilin loop sequence is located within thenon-toxic Pseudomonas exotoxin A sequence. In the present invention, aType IV pilin loop sequence refers to the sequence that forms anintrachain disulfide loop at the C-terminus of the pilin. This loopinteracts and binds to receptors on epithelial cells. The presentinvention is based on, in part, the discovery that the Type IV pilinloop sequence within the Pseudomonas exotoxin A sequence is presented innear-native conformation, and can react with receptors on epithelialcells. As a result, the present chimeric protein comprises the Type IVpilin loop sequence which competes for binding to these epithelialcells, and which can reduce adherence of pathogenic microorganismsexpressing the Type IV pilin to the epithelial cells. Therefore, thechimeric protein can be used on its own or in a composition to directlyreduce adherence of pathogenic microorganisms in a host.

[0009] The present invention is also based on, in part, the discoverythat antisera generated against the chimeric proteins of the inventionare also useful in reducing adherence of pathogenic microorganisms(expressing Type IV pilins) in a host. Since the chimeric proteinpresents the Type IV pilin loop in near-native conformation, thechimeric proteins of the invention, when introduced into a host,generate polyclonal antisera that bind to the pilin loop portion of thechimeric proteins. The antisera can also bind to Type IV pilins onpathogenic microorganism, and thus competitively inhibit binding of thepathogenic microorganisms to epithelial cell receptors. Accordingly, thechimeric protein can be used as a vaccine to generate antisera in a hostwhich can result in reduction of both adherence and colonization ofpathogenic microorganisms in the host.

[0010] Furthermore, since the chimeric protein presents the non-toxicPseudomonas exotoxin A sequence in near-native conformation, thechimeric proteins of the invention, when introduced into a host,generate polyclonal antisera that bind to the non-toxic Pseudomonasexotoxin A as well as to the native Pseudomonas exotoxin A. The nativePseudomonas exotoxin A which is secreted by Pseudomonas aeruginosa isknown to cause cell cytotoxicity by entering into cells byreceptor-mediated endocytosis and then, after a series of intracellularprocessing steps, translocate to the cell cytosol and ADP-ribosylateelongation factor 2. This results in the inhibition of protein synthesisand cell death. The antisera generated against the present chimericprotein can bind exotoxin A released from Pseudomonas and can neutralizecell cytotoxicity. Therefore, should small numbers of Pseudomonasovercome the first line of defense (antibodies against the pilin loopsequence preventing colonization), the normal destructive power of theexotoxin A will be neutralized by antibodies generated against thenon-toxic Pseudomonas exotoxin A sequence.

[0011] The chimeric proteins, the chimeric polynucleotides, and thecompositions of the present invention have many other utilities. Forexample, the chimeric proteins and the compositions comprising chimericproteins can be used to in diagnostic tests, such as immunoassays. Suchdiagnostic tests can be used to detect the presence of microorganismsbearing a Type IV pilin loop sequence, such as Pseudomonas aeruginosa,or to determine whether a host has antisera against a Type IV pilin loopdue to an infection. In another example, the chimeric proteins and thecompositions comprising the chimeric proteins can also be used to purifyantibodies against, e.g., the Type IV pilin loop sequence. In anotherexample, the antibodies against the chimeric protein can be used toclone and isolate other related Type IV pilin sequences.

[0012] Accordingly, in one aspect of the invention, the inventionprovides a chimeric protein comprising: a non-toxic Pseudomonas exotoxinA sequence and a Type IV pilin loop sequence, the Type IV pilin loopsequence being located within the non-toxic Pseudomonas exotoxin Asequence, wherein the chimeric protein is capable of reducing adherenceof a microorganism expressing the Type IV pilin loop sequence toepithelial cells, and further wherein the chimeric protein, whenintroduced into a host, is capable of generating polyclonal antiserathat reduce adherence of the microorganism expressing the Type IV pilinloop sequence to the epithelial cells.

[0013] In another aspect, the invention provides a chimeric proteincomprising: (a) a non-toxic Pseudomonas exotoxin A sequence comprisingdomain Ia, domain II, and domain III; and (b) a Type IV pilin loopsequence, wherein the Type IV pilin loop sequence is located betweendomain II and domain III of the non-toxic Pseudomonas exotoxin Asequence.

[0014] In another aspect, the invention provides a polynucleotideencoding a chimeric protein, the chimeric protein comprising: anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A sequence, wherein the chimeric proteinis capable of reducing adherence of a microorganism expressing the TypeIV pilin loop sequence to epithelial cells, and further wherein thechimeric protein, when introduced into a host, is capable of generatingpolyclonal antisera that prevent adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells.

[0015] In another aspect, the invention provides a polynucleotideencoding a chimeric protein, the chimeric protein comprising: (a) anon-toxic Pseudomonas exotoxin A sequence comprising domain Ia, domainII, and domain III; and (b) a Type IV pilin loop sequence, wherein theType IV pilin loop sequence is located between domain II and domain IIIof the non-toxic Pseudomonas exotoxin A sequence.

[0016] In another aspect, the invention provides a compositioncomprising a chimeric protein, the chimeric protein comprising: anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A sequence, wherein the chimeric proteinis capable of reducing adherence of a microorganism expressing the TypeIV pilin loop sequence to epithelial cells, and further wherein thechimeric protein, when introduced into a host, is capable of generatingpolyclonal antisera that prevent adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells.

[0017] In another aspect, the invention provides a method for elicitingan immune response in a host, the method comprising the step ofadministering to the host an immunologically effective amount of acomposition comprising a chimeric protein comprising: a non-toxicPseudomonas exotoxin A sequence and a Type IV pilin loop sequence, theType IV pilin loop sequence being located within the non-toxicPseudomonas exotoxin A sequence, wherein the chimeric protein is capableof reducing adherence of a microorganism expressing the Type IV pilinloop sequence to epithelial cells, and further wherein the chimericprotein, when introduced into the host, is capable of generatingpolyclonal antisera that prevent adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells.

[0018] In another aspect, the invention provides a method of elicitingan immune response in a host, the method comprising the step ofadministering to the host an immunologically effective amount of anexpression cassette comprising a polynucleotide encoding a chimericprotein comprising: a non-toxic Pseudomonas exotoxin A sequence and aType IV pilin loop sequence, the Type IV pilin loop sequence beinglocated within the non-toxic Pseudomonas exotoxin A, wherein thechimeric protein is capable of reducing adherence of a microorganismexpressing the Type IV pilin loop sequence to epithelial cells, andfurther wherein the chimeric protein, when introduced into the host, iscapable of generating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to theepithelial cells.

[0019] In another aspect, the invention provides a method of generatingantibodies specific for a Type IV pilin loop sequence, comprisingintroducing into a host a composition comprising a chimeric proteincomprising a non-toxic Pseudomonas exotoxin A sequence and a Type IVpilin loop sequence, the Type IV pilin loop sequence being locatedwithin the non-toxic Pseudomonas exotoxin A, wherein the chimericprotein is capable of reducing adherence of a microorganism expressingthe Type IV pilin loop sequence to epithelial cells, and further whereinthe chimeric protein, when introduced into the host, is capable ofgenerating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to epithelialcells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1A illustrates in cartoon form the replacement of domain Ibwith the C-terminal loop of pilin. The pilin insert corresponds to thesequence of pilin reported for the PAK strain of P. aeruginosa.

[0021]FIG. 1B illustrates in cartoon form the domain structure of PEfrom Allured et al., Proc. Natl. Acad. Sci. 83:1320-1324 (1986). PE64lacks the loop region of domain Ib. PE64pil includes the insertion ofthe pilin loop (residues 129-142) of the PAK strain of P. aeruginosa.The deletion of glutamic acid 553 (indicated by a dot) removes an activesite residue (Lukac et al., Infect. Immuno. 56(12):3095-8 (1988)) andproduces proteins PE64Δ553 and PE64Δ553pil with no ADP-ribosylatingactivity. The Ib loop is shown in light shading and the pilin loop indarker shading.

[0022]FIG. 2 illustrates SDS PAGE (Panel A and C) and Western blotanalysis (Panel B) of PE proteins and pilin. A. Lanes 1-4 showsubstantially pure PE proteins (4-5 μg of protein was loaded per lane)after MonoQ chromatography. From left to right the proteins loaded were:PE64, PE64pil, PE64Δ553 and PE64Δ553pil. Purified PAK pilin was added tolane 5. B. Lanes 6-10 show the same proteins as A but probed with amonoclonal antibody to the pilin loop. Lane 11 is PE64Δ553pil after gelfiltration chromatography. Standard proteins and their molecular massesin kDa are indicated.

[0023]FIG. 3 illustrates the toxicity of PE64pil compared to PE64. Toassess the effect of introducing a third party loop into PE, we comparedthe toxicity of PE64 (▪) with PE64pil (▴). Increasing concentrations ofeach protein was added to L929 cells and, after an overnight incubation,inhibition of protein synthesis was determined. Results are expressed aspercent control compared to cells receiving no toxin. Error barsrepresent one SD of the mean from triplicate wells.

[0024]FIG. 4 illustrates the interaction of PE64pil and PE64Δ553pil withimmobilized asialo-GM1. (A). Various concentrations of PE64pil or PE64were added to plates coated with asialo-GM1 and binding was determinedby reactivity with rabbit anti-PE followed by a peroxidase labeled goatanti-rabbit IgG antibody. Absorbance at 450 nm was used to monitorbinding. (B). and (C). To investigate ganglioside specificity, acompetition assay was devised whereby soluble asialo-GM1 ormonosialo-GM1 at 2 ug/ml was preincubated with PE64pil (B) orPE64Δ553pil (C) and the percent residual binding determined as describedin panel (A). For (B) and (C), graphs show the mean of a representativetriplicate experiment. Error bars represent one SD. N.A.=no addition ofcompetitor.

[0025]FIG. 5 illustrates adhesion of Ps. aeruginosa (PAK strain) to A549cells. Bacteria were added to cells at an MOI of 100 in the presence orabsence of potential inhibitors. Peptides were added to a finalconcentration of 40 μM, while proteins were added to a concentration of2 μM. The graph indicates the percentage of cell-bound bacteria comparedto samples with no inhibitor. Error bars represent one standarddeviation from the mean of three independent experiments.

[0026]FIG. 6 illustrates antibody titers post immunization with PE64pilwith and without adjuvant. Sera were collected from each of four rabbits(numbered 87-90) at various times, diluted 1:100 and then added tostreptavidin-coated plates that had been loaded with biotinylated pilinpeptides. Rabbit IgG was detected by the addition of a peroxidaseconjugated goat anti-rabbit antibody. Rabbits 87 and 88 receivedadjuvant while rabbits 89 and 90 did not.

[0027]FIG. 7 illustrates antibody-mediated interference with adhesion toA549 cells. (A). The PAK strain of Ps. aeruginosa was incubated with1:20 to 1:100 dilutions of prebleed or immune (taken after the fourthinjection of antigen) sera from rabbit #87. Bacteria were then added tocells and the percent adhesion determined by comparison with bacteriathat had been incubated in media alone. (B). A 1:20 dilution of serafrom each rabbit, prebleed and immune, was tested for antibody mediatedinterference. (C). Various strains of Ps. aeruginosa were incubated withimmune sera (1:20) from one of the rabbits that received antigen alone(rabbit #90) and one that received antigen plus adjuvant (rabbit #88).For each panel of FIG. 7, the bar represents the number of bacteria percell determined by examining one hundred A549 cells. The error barsrepresent one standard deviation from the mean of three independentexperiments.

[0028]FIG. 8 illustrates antibody-mediated neutralization of PEtoxicity. Immune sera (▴) or prebleed sera (▪) were diluted 1:20 andmixed with PE64 at 1.0 ug/ml. Samples were then diluted to theconcentration indicated and added to L929 cells for an overnightincubation. Results are expressed as percent control of proteinsynthesis compared to cells receiving no toxin. Error bars represent oneSD of the mean from triplicate wells.

DEFINITIONS

[0029] “Pseudomonas exotoxin A” or “PE” is secreted by P. aeruginosa asa 67 kDa protein composed of three prominent globular domains (Ia, II,and III) and one small subdomain (Ib) connecting domains II and III.(Allured et. al., Proc. Natl. Acad. Sci. 83:1320-1324 (1986).) Domain Iaof PE located at the N-terminus and mediates cell binding. In nature,domain Ia binds to the low density lipoprotein receptor-related protein(“LRP”), also known as the α2-macroglobulin receptor (“α2-MR”). (Kounnaset al., J. Biol. Chem. 267:12420-23 (1992).) It spans amino acids 1-252.Domain II mediates translocation to the cytosol. It spans amino acids253-364. Domain Ib has no known function. It spans amino acids 365-399.Domain III is responsible for cytotoxicity and includes an endoplasmicreticulum retention sequence. It mediates ADP ribosylation of elongationfactor 2 (“EF2”), which inactivates protein synthesis. It spans aminoacids 400-613. The native Pseudomonas aeruginosa exotoxin A nucleic acidsequence and the amino acid sequence are shown as SEQ ID NO:1 and SEQ IDNO:2, respectively. SEQ ID NOS: 1 and 2 are the mature form of exotoxinA, wherein the signal sequence has been cleaved off. As a virulencefactor, PE can kill PMNs, macrophages and other elements of the immunesystem (Pollack et al., Infect. Immuno. 19(3):1092-6 (1978)).

[0030] As used herein, “Pseudomonas exotoxin A” or “PE” refer to thosehaving the functions described above and includes the native Pseudomonasexotoxin A having the nucleic acid and amino acid sequences (as shown asSEQ ID NO:1 and SEQ ID NO:2, respectively) and also polymorphicvariants, alleles, mutants and interspecies homologs that: (1) haveabout 80% amino acid sequence identity, preferably about 85-90% aminoacid sequence identity to SEQ ID NO:2 over a window of about 25 aminoacids, preferably over a window of about 50-100 amino acids; (2) bind toantibodies raised against an immunogen comprising an amino acid sequenceof SEQ ID NO:2 and conservatively modified variants thereof; or (3)specifically hybridize (with a size of at least about 500, preferably atleast about 900 nucleotides) under stringent hybridization conditions toa sequence SEQ ID NO:1 and conservatively modified variants thereof. Forexample, genetically modified forms of PE are described in, e.g., Pastanet al., U.S. Pat. No. 5,602,095; Pastan et al., U.S. Pat. No. 5,512,658and Pastan et al., U.S. Pat. No. 5,458,878. Allelic forms of PE areincluded in this definition. See, e.g., Vasil et al., Infect. Immunol.52:538-48 (1986).

[0031] “Non-toxic Pseudomonas exotoxin A” or “non-toxic PE” refers toany Pseudomonas exotoxin A described herein (including modifiedvariants) that lacks ADP ribosylation activity. The ribosylatingactivity of PE is located between about amino acids 400 and 600 of PE.For example, deleting amino acid E553 (“ΔE553”) from domain IIIdetoxifies the molecule. This detoxified PE is referred to as “PEΔE553.” In another example, substitution of histidine residue of PE at426 with a tyrosine residue also inactivates the ADP-ribosylation of PE(see Kessler & Galloway, J. Biol. Chem. 267:19107-11 (1992)). Otheramino acids within domain III can be modified by, e.g., deletion,substitution or addition of amino acid residues, to eliminate ADPribosylation activity. Domain III of non-toxic PE is sometimes referredto herein as “detoxified domain III.”

[0032] The term “a non-toxic Pseudomonas exotoxin A sequence” is usedgenerically to refer to either a nucleic acid sequence or an amino acidsequence of non-toxic Pseudomonas exotoxin A. As used herein, anon-toxic Pseudomonas exotoxin A sequence may be a full length sequenceor portion(s) of the full length sequence. Generally, a non-toxicPseudomonas exotoxin A sequence has one or more domains or portions ofdomains with certain biological activities of a non-toxic Pseudomonasexotoxin A, such as a cell recognition domain, a translocation domain,or an endoplasmic reticulum retention domain. For example, a non-toxicPseudomonas exotoxin A sequence may include only domain II anddetoxified domain III. In another example, a non-toxic Pseudomonasexotoxin A sequence may include only domain Ia, domain II, anddetoxified domain III. In another example, a non-toxic Pseudomonasexotoxin A sequence may include all of domains Ia, Ib, II, anddetoxified III. Therefore, a non-toxic Pseudomonas exotoxin A sequencemay be a contiguous sequence of the native Pseudomonas exotoxin A, or itcan be a sequence comprised of non-contiguous subsequences of the nativePseudomonas exotoxin A that lacks ADP ribosylation activity. While anon-toxic Pseudomonas exotoxin A sequence may be smaller contiguous ornon-contiguous portion(s) of the native PE, the numberings of the nativePE amino acid and nucleic acid sequences are used to refer to certainpositions within the non-toxic Pseudomonas exotoxin A sequence (e.g.,deletion of Glu at position 553).

[0033] A “chimeric protein” or a “chimeric polynucleotide” is anartificially constructed protein or polynucleotide comprisingheterologous amino acid sequences or heterologous nucleic acidsequences, respectively.

[0034] The term “heterologous” when used with reference to a protein ora nucleic acid indicates that the protein or the nucleic acid comprisestwo or more sequences or subsequences which are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid. Forexample, in one embodiment, the nucleic acid has a promoter from onegene arranged to direct the expression of a coding sequence from adifferent gene. Thus, with reference to the coding sequence, thepromoter is heterologous. Similarly, a sequence from a Pseudomonasexotoxin A is heterologous with reference to a Type IV pilin loopsequence when the two sequences are placed in a relationship other thanthe naturally occurring relationship of the nucleic acids in the genome.

[0035] “Type IV pili” refers to filamentous structures covering manygram-negative bacteria, yeast and other microorganisms. The pili on thesurface of a microorganism adhere to epithelial cells. In particular,the pili of Pseudomonas or Candida bind to epithelial cells throughspecific interaction with asialoGM1 receptors. Type IV pili areprimarily composed of protein pilins, which are polymers arranged in ahelical bundle. For example, pili of Pseudomonas aeruginosa have anaverage length of 2.5 μm and consist of a single protein with amolecular mass of around 15,000 (Paranchych et al., Am. Soc. Microbio.343-351 (1990)).

[0036] The term “Type IV pilin” as used herein refer to pilins thatcontain a conserved amino terminal hydrophobic domain beginning with anamino-terminal phenylalanine that is methylated upon processing andsecretion of the pilin. Another characteristic feature of Type IV pilinsis that in the propilin form they contain similar six- or seven-aminoacid long leader peptides, which are much shorter than typical signalsequences. Type IV pilins are expressed by several bacterial genuses,including Neisseria, Moraxella, Bacteroides, Pasteurella andPseudomonas, E. coli, and yeast such as Candida. Species within thesegenuses which express Type IV pilins are, for example, P. aeruginosa, N.gonorrhoeae, N. meningtidis, Pasteurella multocida, M. bovis, B.nodosus. As used herein, the term “Type IV pilin” also includes the Tcppilin of Vibrio, (e.g., V. cholera), that is highly homologous to theType IV pilins of other genuses. Tcp pilin contains the characteristicamino-terminal hydrophobic domain as well as having a modifiedN-terminal amino acid that in this case may be a modified methioninebecause the Tcp pilin gene encodes a methionine residue at the positionwhere all the others encode a phenylalanine. Precursor TcpA contains amuch longer leader sequence than typical Type IV propilins but retainshomology in the region surrounding the processing site. Generally, apilin protein comprises a region at the N-terminus that is highlyconserved, with the rest of the protein containing moderately conservedand hypervariable regions (Paranachych et al., supra). A characteristicfeature of all pilins is an intrachain disulfide loop at the C-terminusof the pilin.

[0037] The amino acid sequences and nucleic acid sequences of Type IVpilins of various microorganisms are known in the art. See, e.g., NCBIDatabase Accession No. M14849, J02609 for Pseudomonas PAK strain; NCBIDatabase Accession No. AAC60462 for Pseudomonas T2A strain; NCBIDatabase Accession No. M11323 for Pseudomonas PAO strain; NCBI DatabaseAccession No. P17837 for Pseudomonas CD strain; NCBI Database AccessionNo. B31105 for Pseudomonas P1 strain; NCBI Database Accession No. Q53391for Pseudomonas KB7 strain; NCBI Database Accession No. AAC60461 forPseudomonas 577B strain; NCBI Database Accession No. A33105 forPseudomonas K122-4 strain; NCBI Database Accession Nos. Z49820, Z69262,and Z69261 for N. meningtidis; NCBI Database Accession Nos. X66144 andAF043648 for N. gonorrhoeae; NCBI Database Accession Nos. U09807 andX64098 for V. cholera; NCBI Database Accession No. AF154834 forPasteurella multocida.

[0038] A “Type IV pilin loop sequence” refers to the sequence that formsan intrachain disulfide loop at the C-terminus of the pilin. This regionis physically exposed at the tip of the pilus, and interacts withepithelial cell receptors. A Type IV pilin loop sequence as used hereincan refer to a sequence between the two cysteine residues that form anintrachain disulfide loop at the C-terminus of the pilin (i.e.,excluding the cysteine residues), or a sequence that includes bothcysteine residues and amino acids between the two cysteine residues.Depending on whether the site of insertion within non-toxic Pseudomonasexotoxin A sequences has cysteine residues, the Type IV pilin loopsequence with or without the flanking cysteine residues can be used tomake chimeric proteins of the invention. Examples of Type IV pilin loopsequence are shown as SEQ ID NOS: 3 to 20.

[0039] The term “immunogenic fragment thereof” or “immunogenic portionthereof” refers to a polypeptide comprising an epitope that isrecognized by cytotoxic T lymphocytes, helper T lymphocytes or B cells.

[0040] “Polyclonal antisera” refers to sera comprising polyclonalantibodies against an immunogen, which sera is obtained from a hostimmunized with the immunogen (e.g., a chimeric protein of the presentinvention).

[0041] Polyclonal antisera that “reduce adherence” of a microorganismexpressing a Type IV pilin loop sequence refer to polyclonal antiserathat reduce adherence of the microorganism by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 100%, compared to a control. A control can bea prebleed or sera that is not exposed to the chimeric proteins of thepresent invention.

[0042] The term polyclonal antisera that “neutralize cytotoxicity” ofPseudomonas exotoxin A in the context of the present invention refer tothe ability of antisera to reduce the inhibition of protein synthesis byPseudomonas exotoxin A. Typically, polyclonal antisera can reduceinhibition of protein synthesis by Pseudomonas exotoxin A by at leastabout 30%, more typically at least about 50%, more typically at leastabout 80%, even more typically at least about 90%, 95%, or 99% comparedto a control. A control can be a prebleed or sera that is not exposed tothe chimeric proteins of the present invention.

[0043] “Nucleic acid” or “polynucleotide” refers to deoxyribonucleotidesor ribonucleotides and polymers thereof in either single- ordouble-stranded form. The term encompasses nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,which have similar binding properties as the reference nucleic acid, andwhich are metabolized in a manner similar to the reference nucleotides.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0044] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

[0045] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

[0046] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

[0047] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0048] “Conservatively modified variants” apply to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or essentially identical amino acid sequences, orwhere the nucleic acid does not encode an amino acid sequence, toessentially identical sequences. Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine. Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

[0049] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid.

[0050] Conservative substitution tables providing functionally similaramino acids are well known in the art. Such conservatively modifiedvariants are in addition to and do not exclude polymorphic variants,interspecies homologs, and alleles of the invention.

[0051] The following eight groups each contain amino acids that areconservative substitutions for one another:

[0052] 1) Alanine (A), Glycine (G);

[0053] 2) Aspartic acid (D), Glutamic acid (E);

[0054] 3) Asparagine (N), Glutamine (Q);

[0055] 4) Arginine (R), Lysine (K);

[0056] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0057] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0058] 7) Serine (S), Threonine (T); and

[0059] 8) Cysteine (C), Methionine (M)

[0060] (see, e.g., Creighton, Proteins (1984)).

[0061] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0062] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, optionally 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

[0063] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

[0064] An “expression cassette” refers to a polynucleotide moleculecomprising expression control sequences operatively linked to codingsequence(s).

[0065] A “vector” is a replicon in which another polynucleotide segmentis attached, so as to bring about the replication and/or expression ofthe attached segment.

[0066] “Control sequence” refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism; in prokaryotes, such control sequences generally includepromoter, ribosomal binding site, and terminators; in eukaryotes,generally, such control sequences include promoters, terminators and, insome instances, enhancers. The term “control sequences” is intended toinclude, at a minimum, all components whose presence is necessary forexpression, and may also include additional components whose presence isadvantageous, for example, leader sequences.

[0067] “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

[0068] A “ligand” is a compound that specifically binds to a targetmolecule.

[0069] A “receptor” is compound that specifically binds to a ligand.

[0070] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0071] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0072] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

[0073] For preparation of monoclonal or polyclonal antibodies, anytechnique known in the art can be used (see, e.g., Kohler & Milstein,Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy(1985)). Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptidesof this invention. Also, transgenic mice, or other organisms such asother mammals, may be used to express humanized antibodies.Alternatively, phage display technology can be used to identifyantibodies and heteromeric Fab fragments that specifically bind toselected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0074] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to fusion proteins can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with fusion protein and not with individualcomponents of the fusion proteins. This selection may be achieved bysubtracting out antibodies that cross-react with the individualantigens. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

[0075] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes an individual antigen or a portionthereof) or may comprise a variant of such a sequence. Polynucleotidevariants may contain one or more substitutions, additions, deletionsand/or insertions such that the biological activity of the encodedchimeric protein is not diminished, relative to a chimeric proteincomprising native antigens. Variants preferably exhibit at least about70% identity, more preferably at least about 80% identity and mostpreferably at least about 90% identity to a polynucleotide sequence thatencodes a native polypeptide or a portion thereof.

[0076] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95% identity overa specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Optionally, the identity exists over aregion that is at least about 25 to about 50 amino acids or nucleotidesin length, or optionally over a region that is 75-100 amino acids ornucleotides in length.

[0077] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

[0078] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 25 to 500, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

[0079] One example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984)).

[0080] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0081] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

[0082] “Immunogen” refers to an entity that induces antibody productionin the host animal.

[0083] “Vaccine” refers to an agent or composition containing an agenteffective to confer a therapeutic degree of immunity on an organismwhile causing only very low levels of morbidity or mortality. Vaccinesand methods for making vaccines are useful in the study of the immunesystem and in preventing and treating animal or human disease.

[0084] An “immunogenic amount” or “immunologically effective amount” isan amount effective to elicit an immune response in a subject.

[0085] “Substantially pure” or “isolated” means an object species is thepredominant species present (i.e., on a molar basis, more abundant thanany other individual macromolecular species in the composition), and asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50% (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition means that about 80% to 90% or more of the macromolecularspecies present in the composition is the purified species of interest.The object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) if the composition consists essentially of a singlemacromolecular species. Solvent species, small molecules (<500 Daltons),stabilizers (e.g., BSA), and elemental ion species are not consideredmacromolecular species for purposes of this definition.“Naturally-occurring” as applied to an object refers to the fact thatthe object can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring.

[0086] A “host” refers to any animal including human or non-humananimals, such as rodents (e.g., mice or rats), primates, sheep, pigs,guinea pigs, etc.

[0087] “Treatment” refers to prophylactic treatment or therapeutictreatment.

[0088] A “prophylactic” treatment is a treatment administered to a hostwho does not exhibit signs of a disease or exhibits only early signs forthe purpose of decreasing the risk of developing pathology.

[0089] A “therapeutic” treatment is a treatment administered to a hostwho exhibits signs of pathology for the purpose of diminishing oreliminating those signs.

Description of the Specific Embodiments

[0090] I. Chimeric Proteins Comprising a Non-Toxic Pseudomonas Exotoxina Sequence and a Type IV Pilin Loop Sequence

[0091] In one aspect, the invention provides a chimeric proteincomprising: a non-toxic Pseudomonas exotoxin A sequence and a Type IVpilin loop sequence, the Type IV pilin loop sequence being locatedwithin the non-toxic Pseudomonas exotoxin A sequence, wherein thechimeric protein is capable of reducing the adhesion or adherence of amicroorganism expressing the Type IV pilin loop sequence to epithelialcells, and further wherein the chimeric protein, when introduced into ahost, is capable of generating polyclonal antisera that reduce adherenceof the microorganism expressing the Type IV pilin loop sequence to theepithelial cells. In some embodiments, the chimeric proteins of theinvention, when introduced into a host, are also capable of generatingpolyclonal antisera that neutralize cytotoxicity of Pseudomonas exotoxinA. In another aspect, the invention provides a chimeric proteincomprising: (a) a non-toxic Pseudomonas exotoxin A sequence comprisingdomain Ia, domain II, and domain III; and (b) a Type IV pilin loopsequence, wherein the Type IV pilin loop sequence is located betweendomain II and domain III of the non-toxic Pseudomonas exotoxin Asequence. In some embodiments, the chimeric protein comprises anon-toxic Pseudomonas exotoxin A sequence including domains Ia, II, andIII in the native organization structure, except that a Type IV pilinloop sequence, partially or completely, replaces domain Ib and islocated between domain II and domain III. Alternatively or additionally,in some embodiments, the chimeric protein comprises a Type IV pilin loopsequence in domain II, replacing amino acids 265 to 287. The nature ofnon-toxic Pseudomonas exotoxin A sequences, various domains of non-toxicPseudomonas exotoxin A sequences, Type IV pilin loop sequences, andtheir physical relationship within chimeric proteins of the inventionare described in detail below.

[0092] A. Non-toxic Pseudomonas Exotoxin A Sequences

[0093] As described in the Definition section above, Pseudomonasexotoxin A or PE is secreted by Pseudomonas aeruginosa and comprisesthree prominent domains (Ia, II, and III) and one small subdomain (Ib)connecting domains II and III. In nature, domain Ia of PE, spanningamino acids 1-252, mediates cell binding. Domain II, spanning aminoacids 253-364, mediates translocation of the protein to the cytosol.Domain Ib, spanning amino acids 365-399, has no known function. DomainIII, spanning amino acids 400-613, is responsible for cytotoxicity andincludes an endoplasmic reticulum retention sequence. It also containssequences that mediates ADP ribosylation of elongation of factor 2(“EF2”), which inactivates protein synthesis and thus rendering PE to betoxic to cells. Thus, domain Ia or its variant that mediates cellbinding is referred to as “a cell recognition domain.” Domain II or itsvariant that mediates translocation of the proteins to the cytosol isreferred to as “a translocation domain.” Domain III or its variant thatfunctions in translocating the protein from the endosome to theendoplasmic reticulum is referred to as “an endoplasmic reticulumretention domain.”

[0094] A non-toxic Pseudomonas exotoxin A sequence refers to anyPseudomonas exotoxin A sequence that lacks ADP ribosylation activity.Generally, a non-toxic Pseudomonas exotoxin A sequence has one or moredomains or portions of domains with certain biological activities. Forexample, a non-toxic Pseudomonas exotoxin A sequence may comprise atranslocation domain (e.g., domain II of Pseudomonas exotoxin A) and anendoplasmic reticulum domain (e.g., detoxified domain III of Pseudomonasexotoxin A without ADP ribosylation activity). In another example, anon-toxic Pseudomonas exotoxin A sequence may be constructed byeliminating amino acids 1-252 yielding a construct referred to as“PE40”. In another example, a non-toxic Pseudomonas exotoxin A sequencemay be constructed by eliminating amino acids 1-279 yielding a constructreferred to as “PE37.” (See Pastan et al., U.S. Pat. No. 5,602,095.).

[0095] Optionally, a cell recognition domain of Pseudomonas exotoxin A(e.g., domain I) or other cell recognition domains unrelated toPseudomonas exotoxin A can be included in the present chimeric proteins.A cell recognition domain can be linked, directly or indirectly, to therest of the chimeric protein. For example, one can ligate sequencesencoding a cell recognition domain to the 5′ end of non-toxic versionsof PE40 or PE37 constructs, which further comprise a Type IV pilin loopsequence.

[0096] 1. Translocation Domain

[0097] The chimeric proteins of the invention comprise a non-toxicPseudomonas exotoxin A sequence comprising a “PE translocation domain.”The PE translocation domain comprises an amino acid sequence sufficientto effect translocation of chimeric proteins that have been endocytosedby the cell into the cytosol. The amino acid sequence is identical to,or substantially identical to, a sequence selected from domain II of PE.

[0098] The amino acid sequence sufficient to effect translocation can bederived from the translocation domain of native PE. This domain spansamino acids 253-364. The translocation domain can include the entiresequence of domain II. However, the entire sequence is not necessary fortranslocation. For example, the amino acid sequence can minimallycontain, e.g., amino acids 280-344 of domain II of PE. Sequences outsidethis region, i.e., amino acids 253-279 and/or 345-364, can be eliminatedfrom the domain. This domain can also be engineered with substitutionsso long as translocation activity is retained.

[0099] The translocation domain functions as follows. After binding to areceptor on the cell surface, the chimeric proteins enter the cell byendocytosis through clathrin-coated pits. Residues 265 and 287 arecysteines that form a disulfide loop. Once internalized into endosomeshaving an acidic environment, the peptide is cleaved by the proteasefurin between Arg279 and Gly280. Then, the disulfide bond is reduced. Amutation at Arg279 inhibits proteolytic cleavage and subsequenttranslocation to the cytosol. Ogata et al., J. Biol. Chem. 265:20678-85(1990). However, a fragment of PE containing the sequence downstream ofArg279 (called “PE37”) retains substantial ability to translocate to thecytosol. Siegall et al., J. Biol. Chem. 264:14256-61 (1989). Sequencesin domain II beyond amino acid 345 also can be deleted withoutinhibiting translocation. Furthermore, amino acids at positions 339 and343 appear to be necessary for translocation. Siegall et al.,Biochemistry 30:7154-59 (1991).

[0100] Methods for determining the functionality of a translocationdomain are described below in the section on testing.

[0101] 2. ER Retention Domain

[0102] The chimeric protein of the invention can also comprise an aminoacid sequence encoding an “endoplasmic reticulum retention domain” aspart of a non-toxic exotoxin A sequence. The endoplasmic reticulum(“ER”) retention domain functions in translocating the chimeric proteinfrom the endosome to the endoplasmic reticulum, from where it istransported to the cytosol. The ER retention domain is located at theposition of domain III in PE. The ER retention domain comprises an aminoacid sequence that has, at its carboxy terminus, an ER retentionsequence. The ER retention sequence in native PE is REDLK (SEQ IDNO:21). Lysine can be eliminated (i.e., REDL (SEQ ID NO:22)) without adecrease in activity. REDLK (from SEQ ID NO:21) can be replaced withother ER retention sequences, such as KDEL (SEQ ID NO:23), or polymersof these sequences. See Ogata et al., J. Biol. Chem. 265:20678-85(1990); Pastan et al., U.S. Pat. No. 5,458,878; Pastan et al., Annu.Rev. Biochem. 61:331-54 (1992).

[0103] Sequences up-stream of the ER retention sequence can be thenative PE domain III (preferably de-toxified), can be entirelyeliminated, or can be replaced by another amino acid sequence. Ifreplaced by another amino acid sequence, the sequence can, itself, behighly immunogenic or can be slightly immunogenic. Activity of thisdomain can be assessed by testing for translocation of the protein intothe target cell cytosol using the assays described below.

[0104] In native PE, the ER retention sequence is located at the carboxyterminus of domain III. Domain III has two functions in PE. It exhibitsADP-ribosylating activity and directs endocytosed toxin into theendoplasmic reticulum. Eliminating the ER retention sequence from thechimeric protein does not alter the activity of Pseudomonas exotoxin asa superantigen, but does inhibit its utility to elicit an MHC ClassI-dependent cell-mediated immune response.

[0105] The ribosylating activity of PE is located between about aminoacids 400 and 600 of PE. In methods of vaccinating a host using thechimeric proteins of this invention, it is preferable that the proteinbe non-toxic. One method of doing so is by eliminating ADP ribosylationactivity. In this way, the chimeric protein can function as a vector forType IV pilin loop sequences to be processed by the cell and presentedon the cell surface with MHC Class I molecules, rather than as a toxin.ADP ribosylation activity can be eliminated by, for example, deletingamino acid E553 (“ΔE553”) of the native PE. See, e.g., Lukac et al.,Infect. and Immun. 56:3095-3098 (1988). In another example, substitutionof histidine residue of PE at 426 with a tyrosine residue alsoinactivates the ADP-ribosylation of PE (see Kessler & Galloway, supra).Other amino acids in domain III can be modified from the protein toeliminate ADP ribosylation activity. An ER retention sequence isgenerally included at the carboxy-terminus of the chimeric protein.

[0106] In one embodiment, the sequence of the ER retention domain issubstantially identical to the native amino acid sequences of the domainIII, or a fragment of it. In some embodiments, the ER retention domainis domain m of PE.

[0107] In another embodiment, a cell recognition domain is inserted intothe amino acid sequence of the ER retention domain (e.g., into domainIII). For example, the cell recognition domain can be inserted justup-stream of the ER retention sequence, so that the ER retentionsequence is connected directly or within ten amino acids of the carboxyterminus of the cell recognition domain.

[0108] B. Cell Recognition Domain

[0109] Optionally, the chimeric protein of the invention can comprise anamino acid sequence encoding a “cell recognition domain.” The cellrecognition domain functions as a ligand for a cell surface receptor. Itmediates binding of the protein to a cell. It can be used to target thechimeric protein to a cell which will transport it to the cytosol forprocessing. A cell recognition domain may not be necessarily included inthe chimeric protein, as a Type IV pilin loop sequence within thechimeric protein targets receptors on epithelial cells.

[0110] The cell recognition domain functions to attach the chimericprotein to a target cell, and it can be any suitable material, e.g., apolypeptide known to a particular receptor in the target cell. Forexample, the cell recognition domain generally has the size of knownpolypeptide ligands, e.g., between about 10 amino acids and about 1500amino acids, or about 100 amino acids and about 300 amino acids. Severalmethods are useful for identifying functional cell recognition domainsfor use in chimeric proteins. One method involves detecting bindingbetween a chimeric protein that comprises the cell recognition domainwith the receptor or with a cell bearing the receptor. Other methodsinvolve detecting entry of the chimeric protein into the cytosol,indicating that the first step, cell binding, was successful. Thesemethods are described in detail below in the section on testing.

[0111] In one embodiment, the cell recognition domain is domain Ia ofPE, thereby targeting the chimeric protein to the α2-MR domain. In otherembodiments domain Ia can be substituted with ligands that bind to cellsurface receptors or antibodies or antibody fragments directed to cellsurface receptors. For example, to target epithelial cells, a cellbinding domain can be a ligand for or antibodies against the EGFreceptor, transferrin receptors, interleukin-2 receptors, interleukin-6receptors, interleukin-8 receptors, or Fc receptors, or poly-IgGreceptors. To target liver cells, a cell binding domain can be, e.g., aligand for or antibodies against asialoglycoprotein receptors. To targetT cells, a cell binding domain can be, e.g., a ligand for or antibodiesagainst CD3, CD4, CD8, or chemokine receptors. To target activatedT-cells and B-cells, a cell binding domain can be, e.g., a ligand for orantibodies against CD25. To target dendritic cells, a cell bindingdomain can be, e.g., ligands for or antibodies against CD11B, CD11C,CD80, and CD86 MHC class I and II. To target macrophages, a cell bindingdomain can be, e.g., ligands for or antibodies against TNFalphareceptors, chemokine receptors, TOLL receptors, M-CSF receptors, GM-CSFreceptors, scavenger receptors, and Fc receptors. To target endothelialcells, a cell binding domain can be, e.g., a ligand for or antibodiesagainst VEGF receptors. Also, cytokine receptors which are found in manycell types can be targeted. Pastan et al. Ann. Rev. Biochem. 61:331-54(1992).

[0112] The cell recognition domain can be located at any suitableposition within the present chimeric proteins. For example, the cellrecognition domain can be located in the N-terminus of the chimericprotein (e.g., position equivalent to domain Ia of non-toxic PE).However, this domain can be moved out of the normal organizationalsequence of exotoxin A. More particularly, the cell recognition domaincan be inserted upstream of the ER retention domain. Alternatively thecell recognition domain can be chemically coupled to the rest of thechimeric protein. Also, the chimeric protein can include a first cellrecognition domain at the location of the Ia domain and a second cellrecognition domain upstream of the ER retention domain. Such constructscan bind to more than one cell type. See, e.g., Kreitman et al.,Bioconjugate Chem. 3:63-68 (1992). For example, TGFα has been insertedinto domain III just before amino acid 604, i.e., about ten amino acidsfrom the carboxy-terminus. This chimeric protein binds to cells bearingEGF receptor. Pastan et al., U.S. Pat. No. 5,602,095.

[0113] The cell recognition domain can be inserted or attached to therest of the chimeric proteins using any suitable methods. For example,the domain can be attached to the rest of the chimeric protein directlyor indirectly using a linker. The linker can form covalent bonds orhigh-affinity non-covalent bonds. Suitable linkers are well known tothose of ordinary skill in the art. In another example, the cellrecognition domain is expressed as a single chimeric polypeptide from anucleic acid sequence encoding the single contiguous chimeric protein.

[0114] C. Type IV Pilin Loop Sequences

[0115] The chimeric protein also comprises a Type IV pilin loop sequencewithin a non-toxic Pseudomonas exotoxin A sequence. The Type IV pilinloop sequence is generally derived from a sequence that forms anintrachain disulfide loop at the C-terminus of the pilin protein. TheType IV pilin loop sequence allows the chimeric protein to react withasialoGM1 receptors on epithelial cells. This loop is dominated by mainchain residues. Therefore, pilins from several strains bind the samereceptor despite sequence variation and the difference in length (e.g.,for certain Pseudomonas strains, 12 and 17 amino acid loops (or 14 to 19amino acids including flanking cysteine residues)). A Type IV loop pilinsequence comprises at least about 5 amino acid residues, typicallybetween about 10 to 100 amino acids, more typically about 12 to 70 aminoacids, even more typically about 12 to 20 amino acids. Embodiments ofthe invention can have one unit of the Type IV pilin loop sequence ormultiple repeating units (e.g., 2, 3, 4, etc.) of the same or differentType IV pilin loop sequences. In some embodiments, the chimeric proteinscomprise more than one Type IV pilin loop sequences at differentlocations.

[0116] A Type IV pilin loop sequence can be derived from anymicroorganism that adhere to epithelial cells. For example, a Type IVpilin sequence can be derived from bacteria or yeast, such asPseudomonas aeruginosa, Neisseria meningtidis, Neisseria gonorrhoeae,Vibro cholera, Pasteurella multocidam or Candida. Examples of a Type IVpilin sequence are shown as SEQ ID NOS: 3 to 20.

[0117] Type IV pilin sequences from different Pseudomonas aeruginosastrains vary in terms of their sequence as well as their length. SeveralPseudomonas aeruginosa strains have a short pilin loop consisting of 14amino acids (from cysteine 129 to cysteine 142) as shown in Table Ibelow. Other Pseudomonas aeruginosa strains have a long pilin loopconsisting of 19 amino acids (from cysteine 133 to 151) as shown inTable 2 below. TABLE 1 P. aeruginosa strains (with a Type IV pilin loopsequence short pilin loop) (Cysteine 129 to Cysteine 142) PAKCTSDQDEQFIPKGC (SEQ ID NO:3) T2A CTSTQDEMFIPKGC (SEQ ID NO:4) PAO, 90063CKSTQDPMFTPKGC (SEQ ID NO:5) CD, PA103 CTSTQEEMFIPKGC (SEQ ID NO:6)K122-4 CTSNADNKYLPKTC (SEQ ID NO:7) KB7, 82932, 82935 CATTVDAKFRPNGC(SEQ ID NO:8) 1071 CESTQDPMFTPKGC (SEQ ID NO:9)

[0118] TABLE 2 P. aeruginosa strains (with a long pilin Type IV pilinloop sequence loop) (Cysteine 133 to Cysteine 151) 577BCNITKTPTAWKIPNYAPANC (SEQ ID NO:10) 1244, 9D2, P1 CKITKTPTAWKPNYAPANC(SEQ ID NO:11) SBI-N CGITGSPTNWKANYAPANC (SEQ ID NO:12)

[0119] Type IV pilin loop sequences from microorganisms other than P.aeruginosa can also be included in the chimeric proteins of theinvention. Examples of Type IV pilin loop amino acid sequences fromother microorganisms are shown in Table 3 below. TABLE 3 MicroorganismType IV pilin loop sequence Neisseria meningtidisCGLPVARDDTDSATDVKADTTDNINTKHL (Z49820) PSTC (SEQ ID NO:13) Neisseriameningtidis CGQPVTRGAGNAGKADDVTKAGNDNEKIN (Z69262) TKHLPSTC (SEQ IDNO:14) Neisseria meningtidis CGQPVTRAKADADAAGKDTTNIDTKHLPS (Z69261) TC(SEQ ID NO:15) Neisseria gonorrhoeae CGQPVTRTGDNDDTVADAKDGKEIDTKHL(pilE; X66144) PSTC (SEQ ID NO:16) Neisseria gonorrhoeaeCGQPVIKRDAGAKTGADDVKADGNNGINT (pilE; AF043648) KHLPSTC (SEQ ID NO:17)Vibrio cholera CKTLVTSVGDMFPFINVKBGAFAAVADLG (U09807)DFETSVADAATGAGVILKSIAPGSANLNL TNITIIVEKLC (SEQ ID NO:18) Vibrio choleraCKTLITSVGDMFPYIAIKAGGAVALADLG (X64098) DFENSAAAAETGVGVIKSIAPASKNLDLTNITHVEKLC (SEQ ID NO:19) Pasteurella multocida CNGGSEVFPAGFC (AF154834)(SEQ ID NO:20)

[0120] One of skill in the art will recognize that the above describedType IV pilin sequences are merely exemplary and that other Type IVpilin sequences can be readily inserted into the chimeric proteins ofthe present invention. For example, Type IV pilin loop sequencesdescribed in, e.g., U.S. Pat. No. 5,612,036 (Hodges et al.) can also beincorporated into the chimeric proteins of the present invention.

[0121] The Type IV pilin loop sequence can be located at any suitableposition within the chimeric protein of the invention. In oneembodiment, the Type IV pilin sequence is inserted between thetranslocation domain (e.g., domain II of non-toxic exotoxin A) and theER retention domain (e.g., domain III of non-toxic exotoxin A). Inanother embodiment, the chimeric protein has the basic organizationstructure of non-toxic Pseudomonas exotoxin A including domain Ia,domain II, domain Ib, and domain III, except that domain Ib is,partially or completely, replaced by the Type IV pilin loop sequence. Innative Pseudomonas exotoxin A, domain Ib spans amino acids 365 to 399.The native Ib domain is structurally characterized by a disulfide bondbetween two cysteines at positions 372 and 379. Domain Ib is notessential for cell binding, translocation, ER retention or ADPribosylation activity. Therefore, it can be partially or entirelyreplaced by a Type IV pilin loop sequence. For example, a Type IV pilinloop sequence can be inserted between the two cysteines at positions 372and 379, replacing the 6 amino acid residues between the two cysteines.In another embodiment, the Type IV pilin loop sequence can be insertedinto the Ib domain without removing any of the Ib domain sequences. Inanother embodiment, the Type IV pilin loop sequence can be positioned inanother location which forms a cysteine-cysteine disulfide bonded loop,such as amino acids 265-287 of domain II of non-toxic Pseudomonasexotoxin A. In some embodiments, more than one Type IV pilin loopsequences can be inserted into different locations within the chimericprotein.

[0122] Depending on whether the site of insertion within a non-toxicPseudomonas exotoxin A sequence has cysteine residues, a Type IV pilinloop sequence with or without cysteine residues at the N- and C-terminican be used. For example, if the site of insert in the non-toxicPseudomonas exotoxin A sequence does not have cysteine residues, then aType IV pilin loop sequence with cysteine residues at its termini (e.g.,14 amino acids shown in SEQ ID NO:3) can be inserted. In anotherexample, if a Type IV pilin loop sequence is inserted in thecysteine-cysteine loop of the native Ib domain, replacing the six aminoacids between the cysteine residues, then a Type IV pilin loop sequencecan be a sequence without terminal cysteines (e.g., 12 amino acidsbetween the two cysteines shown in SEQ ID NO:3). Therefore, acysteine-cysteine loop can be preferably formed within the chimericprotein of the invention. When the Type IV pilin loop sequence withinthe chimeric protein is presented as a cysteine-cysteine disulfidebonded loop, the Type IV pilin loop structure may stick out from therest of the chimeric protein, where it is available to interact with,e.g., asialoGM1 receptors or with immune system components.

[0123] II. Chimeric Polynucleotides and Expression of Polynucleotides

[0124] A. Polynucleotides Encoding the Chimeric Proteins

[0125] In another aspect, the invention provides polynucleotidesencoding the chimeric proteins of the invention. Suitable amino acidsequences of non-toxic Pseudomonas exotoxin A sequences (e.g.,comprising a translocation domain and an ER retention domain), cellrecognition domains, and Type IV pilin loop sequences and their physicallocations within the present chimeric proteins are described in detailabove. Any polynucleotides that encode these amino acid sequences arewithin the scope of the present invention.

[0126] 1. Identification of Non-Toxic Pseudononas Exotoxin a Sequences

[0127] Polynucleotides that encode non-toxic Pseudomonas exotoxin Aamino acid sequences may be identified, prepared and manipulated usingany of a variety of well established techniques. A nucleotide encodingnative Pseudomonas exotoxin A is shown as SEQ ID NO:1. The practitionercan use this sequence to prepare non-toxic Pseudomonas exotoxin Asequences using various cloning and in vitro amplification methodologiesknown in the art. PCR methods are described in, for example, U.S. Pat.No. 4,683,195; Mullis et al. Cold Spring Harbor Symp. Quant. Biol.51:263 (1987); and Erlich, ed., PCR Technology, (Stockton Press, NY,1989); Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995).These primers can be used, e.g., to amplify either the full lengthsequence, partial sequences or a probe of one to several hundrednucleotides, which is then used to screen cDNA or genomic libraries forrelated nucleic acid sequence homologs. Polynucleotides can also beisolated by screening genomic or cDNA libraries (e.g., Pseudomonasaeruginosa) with probes selected from the sequences of the desiredpolynucleotide under stringent hybridization conditions.

[0128] As an illustration, to clone a Pseudomonas exotoxin A sequencecomprising all of the domains (domain Ia, domain II, domain Ib, anddomain III), the following primers can be used:Forward—GGCCCATATGCACCTGATACCCCAT (SEQ ID NO:24); andReverse—GAATTCAGTTACTTCAGGTCCTCG (SEQ ID NO:25). To clone a Pseudomonasexotoxin A sequence comprising domain II, domain Ib, and domain III, thefollowing primers can be used: Forward—GGCCCATATGGAGGGCGGCAGCCTGGCC (SEQID NO:26); and Reverse—GAATTCAGTTACTTCAGGTCCTCG (SEQ ID NO:27).

[0129] Other Pseudomonas exotoxin A constructs that can be used in theembodiments of the invention are also described in, e.g., U.S. Pat. No.5,602,095 (Pastan et al.). As described in the '095 patent, eliminatingnucleotides encoding amino acids 1-252 yields a construct referred to as“PE40.” Eliminating nucleotides encoding amino acids 1-279 yields aconstruct referred to as “PE37.” Non-toxic versions of these constructs(which lack domain Ia of native exotoxin A) are particularly useful forligating them to sequences encoding heterologous cell recognitiondomains to the 5′ end of these constructs. These constructs canoptionally encode an amino-terminal methionine.

[0130] In addition, Pseudomonas exotoxin A can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for a particular desired application. Means to alterPseudomonas exotoxin A in a manner that does not substantially affectthe functional advantages provided by the PE molecules described hereincan also be used and such resulting molecules are intended to be coveredherein.

[0131] Non-toxic Pseudomonas exotoxin A sequences can be generated fromthese Pseudomonas exotoxin A sequences by modifying portions of domainIII so that they lack ADP ribosylation activity. The ribosylatingactivity of PE is located between about amino acids 400 and 600 ofnative Pseudomonas exotoxin A. For example, deleting amino acid E553(“ΔE553”) from domain III detoxifies the molecule. This detoxified PE isreferred to as “PE ΔE553.” Other amino acids within domain III can bemodified by, e.g., deletion, substitution or addition of amino acidresidues, to eliminate ADP ribosylation activity. For example,substitution of histidine residue of PE at 426 with a tyrosine residuealso inactivates the ADP-ribosylation of PE (see Kessler & Galloway,supra).

[0132] In some embodiments, non-toxic Pseudomonas exotoxin A sequencescan be further modified to accommodate cloning sites for insertion of aType IV pilin loop sequence. For example, a cloning site for the Type IVpilin sequence can be introduced between the nucleotides encoding thecysteines of domain Ib of non-toxic Pseudomonas exotoxin A. For example,a nucleotide sequence encoding a portion of the Ib domain between thecysteine-encoding residues can be removed and replaced with a nucleotidesequence encoding an amino acid sequence and that includes a PstIcloning site. This example is described in detail in the Examplesection. Alternatively, a longer portion of domain Ib or entire domainIb can be removed and replaced with an amino acid sequence and thatincludes cloning site(s).

[0133] The construct can also be engineered to encode a secretorysequence at the amino terminus of the protein. Such constructs areuseful for producing the chimeric proteins in mammalian cells. In vitro,such constructs simplify isolation of the chimeric proteins. In vivo,the constructs are useful as polynucleotide vaccines; cells thatincorporate the construct will express the protein and secrete it whereit can interact with the immune system.

[0134] 2. Identification Type IV Pilin Loop Sequences

[0135] Polynucleotides that encode Type IV pilin loop amino acidsequences may be identified, prepared and manipulated using any of avariety of well-established techniques. Type IV pilin nucleotide andamino acid sequences from various microorganisms are well-known in theart. See, e.g., NCBI Database Accession No. M14849 J02609 forPseudomonas PAK strain; NCBI Database Accession No. AAC60462 forPseudomonas T2A strain; NCBI Database Accession No. M11323 forPseudomonas PAO strain; NCBI Database Accession No. P17837 forPseudomonas CD strain; NCBI Database Accession No. B31105 forPseudomonas P1 strain; NCBI Database Accession No. Q53391 forPseudomonas KB7 strain; NCBI Database Accession No. AAC60461 forPseudomonas 577B strain; NCBI Database Accession No. A33105 forPseudomonas K122-4 strain; NCBI Database Accession Nos. Z49820, Z69262,and Z69261 for N. meningtidis; NCBI Database Accession Nos. X66144 andAF043648 for N. gonorrhoeae; NCBI Database Accession Nos. U09807 andX64098 for V. cholera; NCBI Database Accession No. AF154834 forPasteurella multocida. The practitioners can clone and identify otherpilin nucleotides and amino acid sequences from other microorganismsusing various cloning and in vitro amplification methodologies known inthe art. For example, to clone other pilin loop Pseudomonas strains froma library, primers for amplification from the highly conserved 5′ end ofthe pilin gene and the 3′ end of the neighboring gene(Nicotinate-nucleotide pyrophosphorylase) in the Pseudomonas genome canbe used. Exemplary primers PCR (listed in the 5′ to 3′ direction) forsequencing the pilin genes are as follows: pilATG (26 nc)GAGATATTCATGAAAGCTCAAAAAGG (SEQ ID NO:28); and nadB4 (20 nc)ATCTCCATCGGCACCCTGAC (SEQ ID NO:29); or nadB1 (21 nc)TGGAAGTGGAAGTGGAGAACC (SEQ ID NO:30).

[0136] From these Type IV pilin polynucleotides, the portion that formsthe C-terminal intrachain disulfide loop (i.e., Type IV pilin loop) canbe readily identified visually. Examples of Type IV pilin loop aminoacids are shown as SEQ ID NO:3 to 20 in Tables 1-3 above. Any degeneratenucleotides encoding these and other Type IV pilin loop amino acids canbe used to construct chimeric polynucleotides of the invention. In someembodiments, to facilitate insertion of Type IV pilin loop sequence intoa non-toxic Pseudomonas exotoxin A sequence, 5′ and/or 3′ ends of TypeIV pilin loop nucleotide sequence can be modified to incorporatecohesive ends for cloning sites (e.g., PstI).

[0137] As described above, typically, a Type IV pilin loop sequence isinserted into domain Ib, or can partially or fully replace domain Ib ofnon-toxic Pseudomonas exotoxin A. In some embodiments, a Type IV pilinloop sequence can be inserted into other suitable locations within anon-toxic Pseudomonas exotoxin A sequence. For example, a Type IV pilinloop sequence can be inserted in another location of non-toxicPseudomonas exotoxin A which forms a cysteine-cysteine disulfide bondedloop, such as amino acids 265-287 of domain II of non-toxic Pseudomonasexotoxin A. Other suitable locations for insertion can be readily testedusing functional tests described herein. In some embodiments, more thanone Type IV pilin loop sequences can be inserted into chimericpolynucleotides of the invention (e.g., a first pilin loop sequence indomain Ib and a second pilin loop sequence in domain II).

[0138] 3. Identification of Cell Recognition Domain

[0139] Polynucleotides encoding various cell recognition domains arewell-known in the art. As described above, in one embodiment, the cellrecognition domain is domain Ia of PE, thereby targeting the chimericprotein to the α2-MR domain. In this embodiment, the cell recognitiondomain can be readily included in the chimeric polynucleotides using SEQID NO:1 as described above. In other embodiments domain Ia can besubstituted with ligands that bind to cell surface receptors orantibodies or antibody fragments directed to cell surface receptors.Suitable ligands and antibodies or antibody fragments are describedabove in section IB above. Suitable locations for insertion of cellrecognition domain into chimeric proteins and chimeric polynucleotidesare also described above in section IB.

[0140] The cell recognition domain can be inserted or attached to therest of the chimeric proteins using any suitable methods. For example,the domain can be attached to the rest of the chimeric protein directlyor indirectly using a linker. The linker can form covalent bonds orhigh-affinity non-covalent bonds. Suitable linkers are well known tothose of ordinary skill in the art. In another example, the cellrecognition domain is expressed as a single chimeric polypeptide from anucleic acid sequence encoding the single contiguous chimeric protein.

[0141] B. Expression Cassettes and Vectors

[0142] Embodiments of the invention also provide expression cassettesand vectors for expressing the present chimeric proteins. Expressioncassettes are recombinant polynucleotide molecules comprising expressioncontrol sequences operatively linked to a polynucleotide encoding thechimeric protein. Expression vectors comprise these expression cassettesin addition to other sequences necessary for replication in cells.

[0143] Expression vectors can be adapted for function in prokaryotes oreukaryotes by inclusion of appropriate promoters, replication sequences,markers, etc. for transcription and translation of mRNA. Theconstruction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques alsowell known in the art. Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,(Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc.). Useful promoters for suchpurposes include a metallothionein promoter, a constitutive adenovirusmajor late promoter, a dexamethasone-inducible MMTV promoter, a SV40promoter, a MRP polIII promoter, a constitutive MPSV promoter, atetracycline-inducible CMV promoter (such as the human immediate-earlyCMV promoter), and a constitutive CMV promoter. A plasmid useful forgene therapy can comprise other functional elements, such as selectablemarkers, identification regions, and other genes.

[0144] Expression vectors useful in this invention depend on theirintended use. Such expression vectors must contain expression andreplication signals compatible with the host cell. Expression vectorsuseful for expressing the chimeric proteins include viral vectors suchas retroviruses, adenoviruses and adeno-associated viruses, plasmidvectors, cosmids, and the like. Viral and plasmid vectors are preferredfor transfecting mammalian cells. The expression vector pcDNA1(Invitrogen, San Diego, Calif.), in which the expression controlsequence comprises the CMV promoter, provides good rates of transfectionand expression. Adeno-associated viral vectors are useful in the genetherapy methods of this invention.

[0145] A variety of means are available for delivering polynucleotidesto cells including, for example, direct uptake of the molecule by a cellfrom solution, facilitated uptake through lipofection (e.g., liposomesor immunoliposomes), particle-mediated transfection, and intracellularexpression from an expression cassette having an expression controlsequence operably linked to a nucleotide sequence that encodes theinhibitory polynucleotide. See also Inouye et al., U.S. Pat. No.5,272,065; Methods in Enzymology, vol. 185, Academic Press, Inc., SanDiego, Calif. (D.V. Goeddel, ed.) (1990) or M. Krieger, Gene Transferand Expression—A Laboratory Manual, Stockton Press, New York, N.Y.,(1990). Recombinant DNA expression plasmids can also be used to preparethe polynucleotides of the invention for delivery by means other than bygene therapy, although it may be more economical to make shortoligonucleotides by in vitro chemical synthesis.

[0146] The construct can also contain a tag to simplify isolation of theprotein. For example, a polyhistidine tag of, e.g., six histidineresidues, can be incorporated at the amino terminal end of the protein.The polyhistidine tag allows convenient isolation of the protein in asingle step by nickel-chelate chromatography.

[0147] C. Recombinant Cells

[0148] The invention also provides recombinant cells comprising anexpression cassette or vectors for expression of the nucleotidesequences encoding a chimeric protein of this invention. Host cells canbe selected for high levels of expression in order to purify theprotein. The cells can be prokaryotic cells, such as E. coli, oreukaryotic cells. Useful eukaryotic cells include yeast and mammaliancells. The cell can be, e.g., a recombinant cell in culture or a cell invivo.

[0149]E. coli has been successfully used to produce the chimericproteins of the present invention. The protein can fold and disulfidebonds can form in this cell.

[0150] D. Chimeric Protein Purification and Preparation

[0151] Once a recombinant chimeric protein is expressed, it can beidentified by assays based on the physical or functional properties ofthe product, including radioactive labeling of the product followed byanalysis by gel electrophoresis, radioimmunoassay, ELISA, bioassays,etc.

[0152] Once the encoded protein is identified, it may be isolated andpurified by standard methods including chromatography (e.g., highperformance liquid chromatography, ion exchange, affinity, and sizingcolumn chromatography), centrifugation, differential solubility, or byany other standard technique for the purification of proteins. See,generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.(1982), Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc. N.Y. (1990). The actual conditionsused will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, etc., and will be apparent to thosehaving skill in the art.

[0153] After biological expression or purification, the chimericproteins may possess a conformation substantially different than thenative conformations of the constituent proteins. In this case, it ishelpful to denature and reduce the chimeric protein and then to causethe protein to re-fold into the preferred conformation. Methods ofreducing and denaturing polypeptides and inducing re-folding are wellknown to those of skill in the art (see Debinski et al., J. Biol. Chem.268:14065-14070 (1993); Kreitman & Pastan, Bioconjug. Chem. 4:581-585(1993); and Buchner et al., Anal. Biochem. 205:263-270 (1992)). Debinskiet al., for example, describe the denaturation and reduction ofinclusion body polypeptides in guanidine-DTE. The polypeptide is thenrefolded in a redox buffer containing oxidized glutathione andL-arginine.

[0154] E. Testing Functional Properties of the Chimeric Protein

[0155] The functional properties of the chimeric protein as a whole oreach component thereof are using various routine assays. For example,the chimeric proteins are tested in terms of cell recognition, cytosolictranslocation, Type IV pilin adhesion, and immunogenicity. The entirechimeric protein can be tested, or the function of various domains canbe tested by substituting them for native domains of the wild-typeexotoxin A.

[0156]1. Receptor Binding/Cell Recognition

[0157] To determine whether the cell binding domain present in thechimeric protein functions properly, the ability of the chimeric proteinto bind to the target receptor (either isolated or on the cell surface)is tested using various methods known in the art.

[0158] In one method, binding of the chimeric protein to a target isperformed by affinity chromatography. For example, the chimeric proteinis attached to a matrix in an affinity column, and binding of thereceptor to the matrix detected. Alternatively, the target receptor isattached to a matrix in an affinity column, and binding of the chimericprotein to the matrix is detected.

[0159] Binding of the chimeric protein to receptors on cells can betested by, for example, labeling the chimeric protein and detecting itsbinding to cells by, e.g., fluorescent cell sorting, autoradiography,etc.

[0160] In some embodiments, toxic version of chimeric proteins (whichhas ADP ribosylation activity) can be used to test whether the cellbinding domain of the chimeric proteins binds to its target receptor.For example, the toxic version of chimeric proteins can be incubatedwith either cells that express the target receptors or cells that do notexpress the target receptors, and cytotoxic effects of the toxic versionof chimeric proteins can be determined (e.g., by measuring inhibition of[³H]leucine incorporation).

[0161] If antibodies have been identified that bind to the ligand fromwhich the cell recognition domain is derived, they are also useful todetect the existence of the cell recognition domain in the chimericprotein by immunoassay, or by competition assay for the cognatereceptor.

[0162] In above testing methods, typically a specific or selectivereaction of the chimeric protein to a target will be at least twicebackground signal or noise and more typically more than 10 to 100 timesbackground.

[0163] These methods are described in detail in, e.g., Kreitman et al.,Proc. Natl. Acad. Sci. U.S.A 87:8291-5 (1990); Siegall et al., Semin.Cancer Biol. 1:345-50 (1990); Siegall et al., Cancer Res. 50:7786-8(1990); FitzGerald et al., J. Cell Biol. 126(6):1533-41 (1995).

[0164]2. Translocation to the Cytosol

[0165] To determine whether the translocation domain and the ERretention domain of the chimeric protein properly functions, the abilityof the chimeric protein to gain access to the cytosol is tested.

[0166] a) Presence in the Cytosol

[0167] In one method, access to the cytosol is determined by detectingthe physical presence of the chimeric protein in the cytosol. Forexample, the chimeric protein can be labeled and the chimeric proteinexposed to the cell. Then, the cytosolic fraction is isolated and theamount of label in the fraction determined. Detecting label in thefraction indicates that the chimera has gained access to the cytosol.This result can be compared with a control, e.g., background noise orsignal. If the detectable label in the cytosolic fraction is at leasttwice background signal or noise and more typically more than 10 to 100times background, then, this result indicates that the chimeric proteinhas gained access to the cytosol.

[0168] b) ADP Ribosylation Activity

[0169] In another method, the ability of the translocation domain and ERretention domain to effect translocation to the cytosol can be testedwith a construct containing a domain III having ADP ribosylationactivity. Briefly, cells are seeded in tissue culture plates and exposedto the toxic version of the chimeric protein containing the modifiedtranslocation domain or ER retention sequence. ADP ribosylation activityis determined as a function of inhibition of protein synthesis by, e.g.,monitoring the incorporation of ³H-leucine. This method is furtherdescribed in detail in FitzGerald at al., J. Bio. Chem. 273:9951-9958(1998). The incorporation of ³H-leucine in cells exposed the toxicversion of the chimeric protein can be compared to that of a non-toxiccounterpart or to background noise. If the incorporation of ³H-leucinein cells exposed the toxic version is reduced by at least twice, moretypically more than 10 to 100 times that of the non-toxic counterpart(or compared to background noise), then it can be said that the chimericprotein has properly gained entry to the cytosol.

[0170] 3. Type IV Pilin Loop Adhesion

[0171] If the Type IV pilin sequence within the chimeric protein has astructure that is exposed to a solvent and has near-native conformation,the Type IV pilin loop sequence within the chimeric protein should bindto, e.g., asialoGM1 receptors or other receptors on epithelial cells andalso compete with microorganisms expressing the Type IV pilin loopsequence for binding to these receptors. Therefore, whether or not theType IV pilin loop sequence is properly functioning within the chimericprotein is tested by measuring its ability to adhere to epithelial cellsor its ability to block adherence of microorganisms expressing a Type IVpilin loop sequence (e.g., P. aeruginosa) to epithelial cells. Theseassays can be readily designed by one of skill in the art.

[0172] As an example, if a Type IV pilin loop sequence is derived fromP. aeruginosa or Candida, an adhesion assay can be performed with asubstrate coated with asialoGM1. Various concentrations of the chimericprotein comprising Type IV pilin sequence can be assayed for reactivitywith immobilized asialoGM1. To determine specificity of this reactivitybetween the chimeric protein and asialoGM1, a competition assay can beperformed. For example, soluble asialoGM1 can be added to interfere thechimeric protein binding to immobilized asialoGM1. This method isdescribed in detail in the example section IIB3. This binding result canbe compared to a control (e.g., the same chimeric protein without thepilin loop insert or with a scrambled pilin loop sequence insert). Ifthe amount of binding of the chimeric protein to immobilized asialo GM1is at least twice, typically about 10 to 100 times greater than thecontrol, then it can be said that the pilin loop insert in the chimericprotein is functioning properly.

[0173] In another example, one can test the ability of the chimericprotein to block binding of microorganisms expressing the Type IV pilinloop sequence to epithelial cells. The selection of epithelial cellsdepends on which microorganism Type IV pilin loop sequence within thechimeric protein is derived from. For instance, if the Type IV pilinloop sequence within the chimeric protein is derived from V. cholera,then intestinal epithelial cells can be used binding assays. If the TypeIV pilin loop sequence within the chimeric protein is derived from N.gonorrhoeae, then epithelial cells of genital urinary system can be usedfor binding assays. If the Type IV pilin loop sequence within thechimeric protein is derived from P. aeruginosa, then lung epithelialcells can be used for binding assays.

[0174] As an illustration, various Pseudomonas aeruginosa strains thatexpress Type IV pilin can be added different to the human lungepithelial cell line, A549, which will result in the binding ofPseudomonas aeruginosa to these cells. Then, the chimeric protein can beadded. If the Type IV pilin sequence within the chimeric protein ispresent in near-native conformation, the chimeric protein would competewith Pseudomonas aeruginosa binding and would result in reduction ofPseudomonas aeruginosa adherence to the epithelial cells. This method isdescribed in detail in the example section III below. The result fromthis competition assay can be compared to the result obtained with acontrol (e.g., the same chimeric protein except without the pilin loopinsert or the same chimeric protein with a scrambled pilin loop sequenceinsert). If the chimeric protein can reduce Pseudomonas binding at leasttwice or typically about 10 to 100 times better than the control, thenit can be said that the pilin loop insert in the chimeric protein isfunctioning properly.

[0175] 4. Immunogenicity

[0176] To determine whether the chimeric protein retains itsimmunogenicity respect to both parts of the chimeric protein (i.e., aType IV pilin loop sequence and a non-toxic Pseudomonas exotoxin Asequence), properties of the antisera raised against the chimericprotein are tested.

[0177] a) Immunogenicity of Type IV Pilin Sequence

[0178] Immunogenicity of a Type IV pilin sequence within the chimericprotein is tested by adhesion test using the antisera raised against thechimeric protein. An animal, such as a mouse or a rabbit, can beimmunized with a composition comprising the chimeric protein asdescribed below in Example section IVA. The post immunization antiserafrom the animal can be obtained and prepared to determine if theantisera can inhibit binding of microorganisms expressing the Type IVpilin sequence to the epithelial cells. For example, Pseudomonasaeruginosa can be added to the epithelial cells, and the amount ofPseudomonas binding to the epithelial cells is determined. Then, thepost immunization antisera can be added to the epithelial cells todetermine if antisera reduce binding of Pseudomonas aeruginosa to theepithelial cells. This assay is described in detail in Example sectionIVB. If the pilin loop sequence within the chimeric protein is presentin near native conformation, then it is expected that antisera raisedagainst the chimeric protein (at a suitable dilution, e.g., 1:10 or1:100) can reduce Pseudomonas binding by at least about 20%, typicallyat least about 30%, more typically at least about 50%.

[0179] b) Toxin Neutralizing Response

[0180] Immunogenicity of a non-toxic Pseudomonas exotoxin A sequencewithin the chimeric protein is tested by using antisera raised againstthe chimeric protein. Specifically, post immunization antisera is testedfor its ability to neutralize cytotoxicity of Pseudomonas exotoxin A.For example, one can test the inhibition of protein synthesis ofpurified Pseudomonas exotoxin A on eukaryotic cells in culture. WhenPseudomonas exotoxin A is added to eukaryotic cells, it reduces orprevents protein synthesis in cells, causing cell cytotoxicity. Todetermine if antisera can reduce or inactivate cell cytotoxicity ofPseudomonas exotoxin A, Pseudomonas exotoxin A can be incubated withantisera containing antibodies directed against the chimeric protein.This incubated mixture is added to cells in culture. Then, the effect ofantisera on the protein synthesis in the cells can be measured (e.g.,monitoring the incorporation of [³H] leucine). This assay is describedin Example section IVC below and also in Ogata at al., J. Biol. Chem.265(33):20678-85 (1990). If the non-toxic exotoxin A sequence within thechimeric protein is present in near-native conformation, then it isexpected that antisera raised against the chimeric protein (at asuitable dilution, e.g., 1:10 or 1:100) can reduce cytotoxicity ofPseudomonas exotoxin A by at least about 30%, typically at least about50%, more typically at least about 70%, 80%, 90%, 95%, or 99% comparedto a control (e.g., addition of purified Pseudomonas exotoxin A withoutantisera).

[0181] III Compositions Comprising Chimeric Proteins or Polynucleotides

[0182] The invention also provides formulations of one or more chimericpolypeptide or polynucleotide compositions disclosed herein inpharmaceutically-acceptable solutions for administration to a cell or ananimal, either alone or in combination with other components.

[0183] A. Compositions Comprising Chimeric Proteins

[0184] The chimeric protein of the invention can be administereddirectly to a subject as a pharmaceutical composition. Administration isby any of the routes normally used for introducing a chimeric proteininto ultimate contact with the tissue to be treated, preferably themucosal membrane and epithelial cells. The compositions comprisingchimeric proteins are administered in any suitable manner, preferablywith pharmaceutically acceptable carriers. Suitable methods ofadministering such modulators are available and well known to those ofskill in the art. Although more than one route can be used to administera particular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

[0185] Pharmaceutical compositions comprising the chimeric proteins ofthe invention may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the polypeptides into preparations whichcan be used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen.

[0186] Pharmaceutically acceptable carriers, diluents, or excipients aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition.Accordingly, there are a wide variety of suitable formulations ofpharmaceutical compositions of the present invention. For example,pharmaceutical compositions can be formulated for topicaladministration, systemic formulations, injections, transmucosaladministration, oral administration, inhalation/nasal administration,rectal or vaginal administrations. Suitable formulations for variousadministration methods are described in, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985.

[0187] Briefly, for topical administration, the proteins may beformulated as solutions, gels, ointments, creams, suspensions, etc.Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration. For injection, theproteins may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. For oral administration, a composition canbe readily formulated by combining the chimeric proteins withpharmaceutically acceptable carriers to enable the chimeric proteins tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like. For administration by inhalation,the chimeric proteins for use according to the present invention areconveniently delivered in the form of an aerosol spray from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Theproteins may also be formulated in rectal or vaginal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0188] Other suitable formulations and administration methods will bereadily apparent to one of skill in the art and can be applied to thepresent invention.

[0189] B. Compositions Comprising Chimeric Polynucleotides

[0190] The invention also provides compositions comprising thepolynucleotides encoding the chimeric proteins (sometimes referred to as“chimeric nucleic acids” or “chimeric polynucleotides”). These nucleicacids can be inserted into any of a number of well-known vectors for thetransfection of target cells or host tissues. For example, nucleic acidsare delivered as DNA plasmids, naked nucleic acid, and nucleic acidcomplexed with a delivery vehicle such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. For a review of genetherapy procedures, see Anderson, Science 256:808-813 (1992); Nabel &Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada atal., in Current Topics in Microbiology and Immunology Doerfler and Böhm(eds) (1995); and Yu at al., Gene Therapy 1:13-26 (1994).

[0191] Methods of non-viral delivery of nucleic acids includelipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in, e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787;and U.S. Pat. No. 4,897,355) and lipofection reagents are soldcommercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutrallipids that are suitable for efficient receptor-recognition lipofectionof polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

[0192] C. Vaccines

[0193] In some preferred embodiments of the present invention, vaccinesare provided. The vaccines will generally comprise one or morepharmaceutical compositions, such as those discussed above, incombination with an immunostimulant. An immunostimulant may be anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccinepreparation is generally described in, for example, Powell & Newman,eds., Vaccine Design (the subunit and adjuvant approach) (1995).Pharmaceutical compositions and vaccines within the scope of the presentinvention may also contain other compounds, which may be biologicallyactive or inactive.

[0194] Any of a variety of immunostimulants may be employed in thevaccines of this invention. For example, an adjuvant may be included.Most adjuvants contain a substance designed to protect the antigen fromrapid catabolism, such as aluminum hydroxide or mineral oil, and astimulator of immune responses, such as lipid A. Suitable adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 and derivatives thereof(SmithKline Beecham, Philadelphia, Pa.); CWS, TDM, Leif, aluminum saltssuch as aluminum hydroxide gel (alum) or aluminum phosphate; salts ofcalcium, iron or zinc; an insoluble suspension of acylated tyrosine;acylated sugars; cationically or anionically derivatizedpolysaccharides; polyphosphazenes; biodegradable microspheres;monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF orinterleukin-2, -7, or -12, may also be used as adjuvants.

[0195] Any suitable carrier known in the art can be employed in thevaccines of the invention, and the type of carrier will vary dependingon the mode of administration. The vaccines can be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. These formulations and administrationmethods are described above, and will not be repeated in this section.

[0196] Pharmaceutical compositions and vaccines of the present inventionmay be presented in unit-dose or multi-dose containers, such as sealedvials. Such containers are preferably hermetically sealed to preservesterility of the formulation until use. In general, formulations can bestored as suspensions, solutions or emulsions in oily or aqueousvehicles. Alternatively, a pharmaceutical composition or vaccine may bestored in a freeze-dried condition requiring only the addition of asterile liquid carrier immediately prior to use.

[0197] D. Effective Dose

[0198] Determination of an effective amount of the chimeric protein forinducing an immune response in a subject is well within the capabilitiesof those skilled in the art, especially in light of the detaileddisclosure provided herein.

[0199] An effective dose can be estimated initially from in vitroassays. For example, a dose can be formulated in animal models toachieve an induction of an immune response using techniques that arewell known in the art. One having ordinary skill in the art couldreadily optimize administration to humans based on animal data. Dosageamount and interval may be adjusted individually. For example, when usedas a vaccine, the polypeptides and/or polynucleotides of the inventionmay be administered in about 1 to 3 doses for a 1-36 week period.Preferably, 3 doses are administered, at intervals of about 3-4 months,and booster vaccinations may be given periodically thereafter. Alternateprotocols may be appropriate for individual patients. A suitable dose isan amount of polypeptide or DNA that, when administered as describedabove, is capable of raising an immune response in an immunized patientsufficient to protect the patient from infections by microorganismsexpressing Type IV pilin sequence for at least 1-2 years. In general,the amount of polypeptide or nucleic acid present in a dose (or producedin situ by the DNA in a dose) ranges from about 1 pg to about 5 mg perkg host, typically from about 10 pg to about 1 mg, and preferably fromabout 100 pg to about 1 μg. Suitable dose range will vary with the sizeof the patient, but will typically range from about 0.1 mL to about 5mL.

[0200] IV. Methods of Eliciting an Immune Response

[0201] The chimeric proteins of the invention are useful in eliciting animmune response in a host. Eliciting a humoral immune response is usefulin the production of antibodies that specifically recognize the Type IVpilin loop sequence or the non-toxic exotoxin A sequence and inimmunization against microorganisms that bear the Type IV pilinsequence.

[0202] A. Prophylactic and Therapeutic Treatments

[0203] The chimeric proteins can include the Type IV pilin loopsequences from various pathogenic microorganisms, including Pseudomonasaeruginosa, Neisseria meningitides, Neisseria gonorrhoeae, Vibrocholera, etc. Accordingly, this invention provides prophylactic andtherapeutic treatments for diseases involving the pathological activityof pathogens bearing the Type IV pilin loop sequences. The methodsinvolve immunizing a subject with non-toxic Pseudomonas exotoxin A basedchimeric proteins bearing the Type IV pilin sequence. The resultingimmune responses mount an attack against the pathogens, themselves. Forexample, if the pathology results from bacterial or yeast infection, theimmune system mounts a response against the pathogens.

[0204] B. Humoral Immune Response

[0205] The chimeric proteins are useful in eliciting the production ofantibodies against the Type IV loop pilin sequence and the non-toxicPseudomonas exotoxin A sequence by a subject. The chimeric proteins areattractive immunogens for making antibodies against the Type IV pilinloop sequences that naturally occur within a cysteine-cysteine loop:Because they contain the Type IV pilin loop sequences within acysteine-cysteine loop, they present the Type IV pilin loop sequence tothe immune system in near-native conformation. The resulting antibodiesgenerally recognize the native antigen better than those raised againstlinearized versions of the Type IV pilin sequence.

[0206] Methods for producing polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purifiedpolypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST,keyhole limpet hemanocyanin, etc.), or a polypeptide incorporated intoan immunization vector, such as a recombinant vaccinia virus (see, U.S.Pat. No. 4,722,848) is mixed with an adjuvant. Animals are immunizedwith the mixture. An animal's immune response to the immunogenicpreparation is monitored by taking test bleeds and determining the titerof reactivity to the polypeptide of interest. When appropriately hightiters of antibody to the immunogen are obtained, blood is collectedfrom the animal and antisera are prepared. Further fractionation of theantisera to enrich for antibodies reactive to the polypeptide isperformed where desired. See, e.g., Coligan, Current Protocols inImmunology Wiley/Greene, N.Y. (1991); and Harlow and Lane, Antibodies: ALaboratory Manual Cold Spring Harbor Press, N.Y. (1989).

[0207] In various embodiments, the antibodies ultimately produced can bemonoclonal antibodies, humanized antibodies, chimeric antibodies orantibody fragments.

[0208] Monoclonal antibodies are prepared from cells secreting thedesired antibody. These antibodies are screened for binding topolypeptides comprising the epitope, or screened for agonistic orantagonistic activity, e.g., activity mediated through the agentcomprising the non-native epitope. In some instances, it is desirable toprepare monoclonal antibodies from various mammalian hosts, such asmice, rodents, primates, humans, etc. Description of techniques forpreparing such monoclonal antibodies are found in, e.g., Stites et al(eds.) Basic and Clinical Immunology (4th ed.) Lange MedicalPublications, Los Altos, Calif., and references cited therein; Harlowand Lane, Supra; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975) Nature 256: 495-497.

[0209] In another embodiment, the antibodies are humanizedimmunoglobulins. Humanized antibodies are made by linking the CDRregions of non-human antibodies to human constant regions by recombinantDNA techniques. See Queen et al., U.S. Pat. No. 5,585,089.

[0210] In another embodiment of the invention, fragments of antibodiesagainst the Type IV pilin loop sequence are provided. Typically, thesefragments exhibit specific binding to the Type IV pilin loop sequencesimilar to that of a complete immunoglobulin. Antibody fragments includeseparate heavy chains, light chains, Fab, Fab′ F(ab′)₂ and Fv. Fragmentsare produced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins.

[0211] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors. See, Huse et al.,Science 246: 1275-1281 (1989); and Ward et al., Nature 341: 544-546(1989).

[0212] An approach for isolating DNA sequences which encode a humanmonoclonal antibody or a binding fragment thereof is by screening a DNAlibrary from human B cells according to the general protocol outlined byHuse at al., Science 246:1275-1281 (1989) and then cloning andamplifying the sequences which encode the antibody (or binding fragment)of the desired specificity. The protocol described by Huse is renderedmore efficient in combination with phage display technology. See, e.g.,Dower at al., WO 91/17271 and McCafferty at al., WO 92/01047. Phagedisplay technology can also be used to mutagenize CDR regions ofantibodies previously shown to have affinity for the polypeptides ofthis invention or their ligands. Antibodies having improved bindingaffinity are selected.

[0213] The antibodies of this invention are useful for affinitychromatography in isolating agents bearing the Type IV pilin sequence.Columns are prepared, e.g., with the antibodies linked to a solidsupport, e.g., particles, such as agarose, Sephadex, or the like, wherea cell lysate is passed through the column, washed, and treated withincreasing concentrations of a mild denaturant, whereby purified agentsare released.

[0214] As described in the Example section, sera from immunized rabbitshad two reactivities: one that blocks adhesion and one that neutralizesexotoxin A. Therefore, by introducing the chimeric protein as acomposition (e.g., a vaccine) into a subject, antibodies that preventcolonization of microorganisms bearing Type IV pilin sequences (e.g.,Pseudomonas aeruginosa) can be provided in the subject. In particularfor Pseudomonas aeruginosa, should small numbers of these bacteriaovercome this defense, the normal destructive power of the exotoxin Awill be also neutralized by the antisera.

[0215] C. IgA-mediated Secretory Immune Response

[0216] Mucosal membranes are primary entryways for many infectiouspathogens, including those bearing the Type IV pilin sequences. Mucosalmembranes include, e.g., the mouth, nose, throat, lung, vagina, rectumand colon. As a defense against entry, the body secretes secretory IgAon the surfaces of mucosal epithelial membranes against pathogens.Furthermore, antigens presented at one mucosal surface can triggerresponses at other mucosal surfaces due to trafficking ofantibody-secreting cells between these mucosae. The structure ofsecretory IgA has been suggested to be crucial for its sustainedresidence and effective function at the luminal surface of a mucosa. Asused herein, “secretory IgA” or “sIgA” refers to a polymeric moleculecomprising two IgA immunoglobulins joined by a J chain and further boundto a secretory component. While mucosal administration of antigens cangenerate an IgG response, parenteral administration of immunogens rarelyproduce strong sIgA responses.

[0217] Pseudomonas exotoxin binds to receptors on mucosal membranes.Therefore, the chimeric proteins comprising non-toxic exotoxin Asequences are an attractive vector for bringing the type IV pilin loopsequence to a mucosal surface. There, the chimeric proteins elicit anIgA-mediated immune response against the chimeric proteins. Accordingly,this invention provides the non-toxic Pseudomonas exotoxin A-basedchimeric proteins comprising a Type IV pilin loop sequence from apathogen that gains entry through mucosal membranes. The cellrecognition domain can be targeted to any mucosal surface receptor.These chimeric proteins are useful for eliciting an IgA-mediatedsecretory immune response against immunogens that gain entry to the bodythrough mucosal surfaces. The chimeric proteins used for this purposeshould have ligands that bind to receptors on mucosal membranes as theircell recognition domains. For example, epidermal growth factor binds tothe epidermal growth factor receptor on mucosal surfaces.

[0218] The chimeric proteins can be applied to the mucosal surface byany of the typical means, including pharmaceutical compositions in theform of liquids or solids, e.g., sprays, ointments, suppositories orerodible polymers impregnated with the immunogen. Administration caninvolve applying the immunogen to a plurality of different mucosalsurfaces in a series of immunizations, e.g., as booster immunizations. Abooster inoculation can also be administered parenterally, e.g.,subcutaneously. The chimeric protein can be administered in doses ofabout 1 μg to 1000 μg, e.g., about 10 μg to 100 μg.

[0219] The IgA response is strongest on mucosal surfaces exposed to theimmunogen. Therefore, in one embodiment, the immunogen is applied to amucosal surface that is likely to be a site of exposure to theparticular pathogen. Accordingly, depending on the site of exposure tothe particular pathogen, the chimeric proteins can be administered tothe lung, nasal mucosa, vaginal, anal or oral mucosal surfaces, or theycan be given as an oral medication. For example, for cystic fibrosispatients, the chimeric proteins can be administered to the lung.

[0220] Mucosal administration of the chimeric protein of this inventionresult in strong memory responses, both for IgA and IgG. Therefore, invaccination with them, it is useful to provide booster doses eithermucosally or parenterally. The memory response can be elicited byadministering a booster dose more than a year after the initial dose.For example, a booster dose can be administered about 12, about 16,about 20 or about 24 months after the initial dose.

[0221] The potential value of a Pseudomonas vaccine relates in part toits ability to protect individuals broadly from the strains that arepresent in the environment. Based on the length of the pilin loopinsert, there are two groupings for Ps. aeruginosa: one group with a 12amino acid sequence and one with a 17 amino acid insert. Both loopsapparently bind asialo-GM1 and are thought to exhibit similarstructures. Reflecting this, we note that our vaccine protein,containing a 12 amino acid loop from the PAK strain, was able togenerate antibodies that were reactive not only for strains with theshorter loop but also for the SBI-N strain, which displayed the longerloop. Our studies have also provided additional sequence data for pilinand pilin loop sequences. We report here two pilin loop sequences (thosefor Ps. aeruginosa strain 1071 and Ps. aeruginosa strain SBI-N) thathave not previously been entered in databases (Tables 1 and 2).

[0222] Chronic pulmonary colonization by Ps. aeruginosa is associatedwith a decline in the clinical course of CF patients. Frequently,antibiotic therapy, even via pulmonary delivery, fails to eradicate Ps.aeruginosa infections in these patients (Steinkamp, G., B. et al.Pediatr Pulmonol 6(2):91-8(1989)). Controlling Ps. aeruginosainfections, or better yet, preventing them, has thus become a criticalunmet medical need in the care of CF patients ((Bauernfeind, A. et al.Behring Inst Mitt(98):256-61 (1997)). To address this, a number ofvaccine approaches have been explored, many focused on outer membraneconstituents (Matthews-Greer, J. M., et al.; J Infect Dis 155(6):1282-91 (1987); Owen, P. Biochem Soc Trans 20(1):1-6 (1992); Sawa, etal.; Nat Med 5(4):392-8(1999), some on toxins (Chen, T. Y., et al. JBiomed Sci 6(5):357-63 (1999); Denis-Mize, K. S., et al.; FEMS ImmunolMed Microbiol 27(2):147-54 (2000); Gilleland, H. E., et al.; J MedMicrobiol 38(2):79-86 (1993); Matsumoto, et al.; J Med Microbiol47(4):303-8 (1988)), and some on a combination approach (Cryz, S. J., etal.; Antibiot Chemother 39:249-55 (1987); Cryz, S. J., et al. InfectImmun 52(1):161-5 (1986); Cryz, S. J., et al.; Infect Immun 55(7):1547-51 (1987); and Cryz, S. J., et al. J Infect Dis 154(4):682-8 (1986)(Johansen, H. K., et al.; APMIS 102(7):545-53(1994).

[0223] The compositions of the present invention in some embodiments areused to treat persons at risk of infection and particularly, Pseudomonasaeruginosa infection. These persons include, in particular, hospitalizedpatients having cystic fibrosis, bum wounds, organ transplants,compromised immune function, or intravenous-drug addition.

[0224] Previously, we compared the subcutaneous route with mucosaldelivery of toxin-V3 loop proteins (Mrsny, R. at al., Vaccine17(11-12):1425-33 (1999). Results of mucosal vaccination indicated thata robust anti-V3 loop response could be achieved with high titerresponses of both serum IgG and secretory IgA antibodies. Because thetoxin-pilin chimeric protein is a candidate vaccine to preventPseudomonas colonization in CF, one embodiment provides a vaccinedelivered to target mucosal antibody responses at airway epithelia.

EXAMPLES

[0225] I. Construction of Plasmids

[0226] Four plasmids, pPE64, pPE64Δ553, pPE64pil, pPE64Δ553pil, wereconstructed. Plasmid pPE64 encodes native the Pseudomonas exotoxin A,except the plasmid encoded a slightly smaller version of PE that lackedmuch of domain Ib and has a novel PstI site in domain Ib as described indetail below. Plasmid pPE64Δ553 encodes the a non-toxic version ofplasmid pPE64, whereby the plasmid pPE64 was modified by subcloning tointroduce the enzymatically inactive domain III of PE (i.e., Glu atamino acid position 553 is deleted). To generate a PE-based pilinchimeric protein, an oligonucleotide duplex that encoded amino acids129-142 from the PAK strain of pilin was synthesized. Then plasmidpPE64pil is constructed based on plasmid pPE64, wherein a pilin loopsequence from P. aeruginosa PAK strain was inserted into the PstI siteof plasmid pPE64. Plasmid pPE64Δ553pil is constructed based on plasmidpPE64Δ553, wherein a pilin loop sequence from P. aeruginosa PAK strainwas inserted into the PstI site of plasmid pPE64Δ553. All of thesevectors were constructed without a bacterial secretion sequence whichallowed recombinant proteins to be expressed as inclusion bodies.

[0227] Specifically, plasmids pPE64 and pPE64Δ553 are constructed asfollows. Plasmid pMOA1A2VK352 (Ogata et al., J. Biol. Chem. 267,25396-401 (1992)), encoding PE, was digested with Sfi1 and ApaI(residues 1143 and 1275, respectively) and then re-ligated with a duplexcontaining a novel Pst1 site. The coding strand of the duplex had thefollowing sequence: 5′-tggccctgac cctggccgcc gccgagagcg agcgcttcgtccggcagggc accggcaacg acgaggccgg cgcggcaaac ctgcagggcc-3′. The resultingplasmid encoded a slightly smaller version of PE and lacked much ofdomain Ib. The Pst1 site was then used to introduce duplexes encodingpilin loop sequences flanked by cysteine residues. To make non-toxicproteins, vectors were modified by the subcloning in an enzymaticallyinactive domain III from pVC45ΔE553. An additional subcloning, from pJH4(Hwang et. al., Cell 48:129-136 (1987)), was needed to produce a vectorthat lacked a signal sequence. Construction of plasmids pPE64 andpPE64Δ553 are also described in FitzGerald et al., J. Biol. Chem.273(16):9951-8 (1998).

[0228] Plasmids pPE64pil and pPE64Δ553pil with a pilin loop sequenceinsert were constructed based on plasmids pPE64 and pPE64Δ553,respectively. A 54 bp sense oligonucleotide with cohesive ends for PstIand encoding the 12 amino acid pilin loop of the PAK strain, wasannealed with a 54 bp antisense oligonucleotide in 10 mM Tris/HCl, 50 mMNaCl pH 7.4. The sense and antisense oligonucleotides had the followingsequences: Sense 5′-TTGTACTAGTGATCAGGATGAACAGTTTATTCCGAAAGGTTGTTCACGTATGCA-3′; Antisense 5′-TACGTGAACAACCTTTCGGAATAAACTGTTCATCCTGATCACTAGTACAATGCA-3′.

[0229] Annealing was accomplished by heating to 94° C. for 5 minfollowed by cooling to 25° C. over a period of 40 min. Plasmids pPE64and pPE64Δ5532), encoding enzymatically active and inactive PErespectively, were digested with PstI at residue 1470. (FitzGerald, D.J., et al., J Biol Chem 273(16):9951-8 (1998). Ligation with thephosphorylated pilin oligoduplex destroyed the PstI site and introduceda unique SpeI site. A XhoI/SpeI double digest was used to check for thecorrect orientation of the insert. Ligation of the pilin oligoduplexinto the PstI-cut vector was followed by several characterization stepsto confirm the presence of the pilin insert in the correct orientation.Final constructs were verified by dideoxy double strand sequencing.

[0230] II. Expression and Characterization of Proteins

[0231] A. Expression and Purification

[0232] Using the T7 expression system described by Studier at al.,(Methods Enzymol. 185:60-89 (1990)), four PE-related proteins wereexpressed E. coli. These included PE64, PE64Δ553, PE64pil andPE64Δ553pil.

[0233] Chimeric proteins were expressed and isolated as inclusion bodiesas described in Buchner et al., Anal. Biochem. 205(2):263-70 (1992).Each protein was expressed separately and purified to near homogeneity.Briefly, strain BL21(λDE3) was transformed with plasmids harboring a T7promoter upstream of the initial ATG of the toxin-expressing vectors.Cultures were grown in Superbroth (KD Medical, Bethesda, Md.) withampicillin (50 ug/ml) and then induced for protein expression by theaddition of IPTG (1 mM). After two hours of further culture, bacterialcells were harvested by centrifugation. Following cell lysis, expressedproteins were recovered in inclusion bodies.

[0234] Proteins were solubilized with Guanidine HCl (6.0 M), 2 mM EDTApH 8.0 plus dithioerythreitol (65 mM). Solubilized proteins were thenrefolded by dilution into a redox shuffling buffer (Buchner et al.,Anal. Biochem. 205(2):263-70 (1992). Refolded proteins were dialyzedagainst a 20 mM Tris, 100 mM urea pH 7.4, adsorbed on Q Sepharose(Amersham Pharmacia Biotech), washed with 150 mM NaCl, 20 mM Tris, 1 mMEDTA pH 6.5 and eluted with 280 mM NaCl, 20 mM Tris, 1 mM EDTA. Elutedproteins were diluted 5-fold and then adsorbed onto a MonoQ column (HR10/10, Amersham Pharmacia Biotech) and further purified by theapplication of a linear salt gradient (0-0.4 M NaCl in Tris EDTA, pH7.4). PE proteins eluted between 0.2 and 0.25 M NaCl. Final purificationwas achieved using a gel filtration column (Superdex 200, AmershamPharmacia Biotech) in PBS, pH 7.4.

[0235] B. Characterization of Proteins

[0236] 1. Western Blot Analysis

[0237] The PK99H mouse monoclonal antibody and purified pilin proteinswere obtained from Dr. Randall Irvin, University of AIberta, Canada.Antimouse IgG and antirabbit IgG antibodies were used to detect primaryantibodies in Western blots and ELISAs (available from Jackson ImmunoResearch Lab, West Grove, Pa.).

[0238] Proteins were initially analyzed by SDS-PAGE (FIG. 2A).Substantially pure proteins were obtained using the purification schemeoutlined above. In Western blot analysis the PE64pil and PE64Δ553pilproteins reacted with PK99H, a monoclonal antibody to the C-terminalloop of pilin (FIG. 2B). The same antibody also reacted with solublepreparations of the same proteins, indicating that the pilin insert wasexposed on the surface of the PE-pilin chimeric protein. PE proteinswithout inserts did not react with the PK99H antibody (FIG. 2B).

[0239] 2. Cytotoxicity Assay

[0240] To investigate the influence of the pilin insert on toxinstructure and function, the two enzymatically active proteins, PE64 andPE64pil, were compared in a cytotoxicity assay. Cytotoxicity assaymethods described in Ogata et al., J. Biol. Chem. 265(33):20678-85(1990) was used. Concentrations of PE64 or PE64pil ranging from 0.002-20ng/ml were added to L929 cells for an overnight incubation. Cytotoxicitywas then determined by measuring the inhibition of cellular proteinsynthesis (e.g., monitoring the incorporation of ³H-leucine). Dataindicated that PE64 and PE64pil exhibited similar toxicities with IC₅₀values in the range of 0.1 ng/ml for both proteins (FIG. 3). This resultsuggested that the insert of 14 amino acids did not unduly perturb toxinfunction and, by inference, toxin structure.

[0241] 3. Reactivity with Immobilized Asialo-GM1

[0242] Previous results had indicated that synthetic peptides derivedfrom the C-terminus of pilin could block the binding of pili toepithelial cells (Irvin at al., Infect. Immun. 57(12):3720-6 (1989); Yu,L. at al., Mol Microbiol 19(5):1107-16 (1996)). Blocking was attributedto peptide binding to asialo-GM1 on the surface of epithelial cells. Totest the functionality of the pilin insert in the PE64 proteins, variousconcentrations of PE64pil were assayed for reactivity with immobilizedasialo-GM1.

[0243] 96-well plates were coated with asialo-GM1 or monosialo-GM1(Sigma Chem Co, St Louis, Mo.) that had been solubilized in methanol. A100 μl solution of ganglioside (5 μg/ml) was added to each well andevaporated at 4° C. until dry. Wells were washed 3 times with PBS andblocked with Fish-gelatin-PBS (BioFX, Randallstown, USA) for 16 h at 4°C. Test proteins in blocking buffer were added at variousconcentrations. After incubation for 1 h at 22° C., the supernatant wasremoved and bound protein was detected using heat-inactivated antiPE64Δ553pil serum (1:100) as the primary antibody. For competitionstudies, proteins at 0.2 ug/mI were incubated with 2 ug/ml of asialo-GM1or monosialo-GM1 for 30 min at room temperature. Samples were then addedto asialo-GM1 coated plates as above.

[0244] Increasing concentrations of PE64pil from 0.1-2.0 ug/ml reactedspecifically with immobilized asialo-GM1 (FIG. 4A). PE64 was used as acontrol and exhibited only a low level of binding (FIG. 4A). Additionalstudies were carried out to confirm the ganglioside specificity of bothPE64pil and PE64Δ553pil. Soluble asialo-GM1 reduced the binding ofPE64pil and PE64Δ553pil to immobilized asialo-GM1 while the addition ofmonosialo-GM1 did not (FIGS. 4B and 4C). Neither ganglioside interferedwith the low level binding of PE64 and PE64Δ553 (FIGS. 4B and 4C). Takentogether, these results confirmed not only the presence of reactivepilin sequences but revealed a gain-of-function for the PE64pilproteins.

[0245] III. Adhesion Assays

[0246] A. Pseudomonas Strains

[0247] The following strains of Pseudomonas were used in adhesion andother assays: PAK, PAO1, SBN-1, 1071, M2, 82932, 82935 and 90063.Pseudomonas strains used for adherence studies were grown on LB agar andthen in M9 minimal medium (KD Medical, Bethesda, Md.) supplemented with0.4% glucose at 30° C. without shaking. Cultures in late log phase wereroutinely used for adhesion assays.

[0248] B. Cell Cultures

[0249] A549 (ATCC, CCL-185), L929 (ATCC, CCL-1), WI 38, Vero and CHOcells were maintained in DMEM/F12 or RMPI 1640 supplemented with 10%fetal bovine serum (FBS), 2.5 mM glutamine, standardPenicillin/Streptomycin (100 U/100 ug/ml, GibcoBRL, Grand Island, USA)(further designed as complete medium) in 5% CO₂ at 37° C. Cells were fedevery 2 to 3 days and passaged every 5 to 7 days. For assays, cells wereseeded into 24-well or 96-well plates and grown to confluence.

[0250] C. Quantification of Bacterial Adherence

[0251] To quantify the association of Pseudomonas with A549 cells, wefollowed the adhesion assay described by Chi et al., Infect. Immun.59(3):822-8 (1991). Briefly, A549 cells were grown in a 24 well plates(antibiotic free medium), to a density of approximately 2×10⁴ cells perwell. Cells were washed three times in HBSS without serum and wereoverlayed with 0.5 ml of DMEM/F12 complete medium without FBS. A MOI of20 was achieved by adding 10 μl of an appropriate bacterial dilution.Plates were incubated for 1 or 2 h at 37° C., 5% CO₂.

[0252] To remove unbound bacteria, cells were gently washed three timeswith HBSS. Cells were then fixed for 1 h in 3.7% paraformaldehyde, 200mM HEPES, pH 7.2. Cells were washed twice with saline and stained with10% Giemsa for 10 min. Samples were washed three times with water andexamined under light microscopy at 400× magnification. Adherent bacteriawere quantified by counting the cell-associated bacteria of one hundredA549 cells.

[0253] D. Results

[0254] Pilin-mediated adhesion to epithelial cells allows P. aeruginosato initiate an infection. Agents that block adherence will thereforereduce the bacterial burden. The following three peptides weresynthesized: a long C-terminal peptide (peptide 1:acetyl-KCTSDQDEQFIPKGCSK-NH₂) corresponding to amino acids 128-142 ofthe PAK strain (this peptide was oxidized to allow the formation of adisulfide bond), a core peptide (peptide 2: acetyl-DEQFIPK-NH₂)corresponding to amino acids 134-140 and a scrambled peptide (peptide 3:acetyl-QIDPEFK-NH₂) having the same amino acid composition as the corebut in a jumbled sequence. To enhance stability, the N-termini of thesesynthetic peptides were acetylated while the C-termini were amidated.These peptides were custom synthesized by Sigma Genosys. The samepeptides were also synthesized with a biotin label.

[0255] To test these peptides functionally, an adhesion assay wasdevised whereby washed bacteria of P. aeruginosa PAK strain were addedto the human lung epithelial cell line, A549. Specifically, cultures ofconfluent A549 cells were incubated 60 min at 37° C. with 40 μM peptide1, 40 μM peptide 2, 40 μM peptide 3, 2 nmol/ml PAK-pilin protein, 2nmol/ml PE64, 2 nmol/ml PE64Δ553, 2 nmol/ml PE64pil, 2 nmol/mlPE64Δ553pil and 4 nmol/ml bovine albumin. Washed once with prewarmedDMEM and P. aeruginosa PAK strain was added at a MOI of approximately 50in DMEM, 2% FBS. Bacteria were centrifuged onto the cells (700 g, 5 min)and incubated 60 min, 37° C. 5% CO₂. Adherence was determined asdescribed above.

[0256] The results were as follows. Adherence to A549 cells was reducedby approximately 50% in the presence of 40 μM of the long or the corepilin peptide (see FIG. 5). The scrambled peptide did not interfere withadherence.

[0257] Because the PE-pilin proteins had exhibited binding activity toasialo-GM1, these were also tested. At approximately the same molarconcentration as the synthetic peptides, PE64pil and PE64Δ553pil alsoblocked bacterial adherence. Effects were due to the presence of theinsert, because toxin molecules without insert failed to compete foradherence.

[0258] IV. Immune Response to PE64Δ553PIL

[0259] A. Production of Polyclonal Antibodies

[0260] To test the ability of the toxin-pilin protein to generaterelevant antibody responses, four rabbits were injected with thePE64Δ553pil protein. Two rabbits (numbered 87 and 88) received theprotein plus adjuvant (complete Freunds for the first injection followedby incomplete Freunds for subsequent injections) and two (numbered 89and 90) received the protein alone. Two hundred micrograms of proteinper injection was given subcutaneously for a total of four cycles spacedapproximately 2 weeks apart. About 12 ml serum was isolated biweeklyfrom each rabbit. The sera were heat inactivated to 20 min, 56° C. anddilutions thereof were used for assays without further purification.Anti-pilin titers were determined using an ELISA assay wherebiotinylated pilin peptides were immobilized on strepavidin coatedplates. Over the period of immunization, anti-pilin titers increased inall four animals (FIG. 6). However, the speed and extent of the responsewere greater in the two rabbits that received antigen plus adjuvant. Toavoid complement-mediated bacterial killing, immune sera were heatinactivated. This treatment did not significantly alter antibody titersin the ELISA assay (data not shown).

[0261] B. Inhibition of Adhesion by Post Immunization Sera

[0262] To assess antibody mediated inhibition of adherence,anti-PE64Δ553pil rabbit sera were incubated at dilutions from 1:20 to1:100 with 4×10⁵ bacteria at 22° C. for 30 min. Bacteria were thencentrifuged, resuspended in DMEM without supplements and added toconfluent monolayers of A549 cells at a MOI of 20 for 1-2 hrs. Adherencewas determined as described above. Immune sera taken after the fourthinjection were compared to prebleed samples taken from the same rabbits.

[0263] 1. Inhibition of P. aeruginosa (PAK Strain)

[0264] Sera taken 2 weeks after the last injection were assayed forblocking activity in the bacterial adherence assay. Compared toprebleeds, immune sera at various dilutions blocked adherence of the PAKstrain of Ps. aeruginosa (FIG. 7A). Reduction of adherence ranged from60% at a dilution of 1:100 to 90% at a dilution of 1:20. At a dilutionof 1:20, blocking activity was comparable without regard to the presenceof adjuvant in the antigen preparation (FIG. 7B).

[0265] 2. Inhibition of P. aeruginosa (Various Strains)

[0266] Inhibition of PAK strain adhesion confirmed that rabbitsresponded to the specific pilin sequence that was administered in thevaccine. However, because the C-terminal loop of pilin exhibitsconsiderable sequence variation, it was important to determine thereactivity of the immune sera for other strains of Ps. aeruginosa.Strains PAO1, 1071, SBI-N, 82935, 82932, 90063 1244 and M2 were testedfor adherence to A549 cells under similar conditions as the PAK strain.The specific cell binding of all strains were reduced in adhesion whenheat inactivated immune rabbit sera were mixed with bacteria at a 1:20dilution (FIG. 7C). The reduction in adhesion among the differentstrains was more or less in the range of the PAK strain (about 90%reduction).

[0267] While it was unlikely that each of the above strains expressedthe same loop sequence as the PAK strain, it was of interest to analyzevariations at this portion of the pilin gene. Pilin sequences weredetermined by generating PCR clones of each strain's pilin gene andsequencing these. Primers for amplification were from the 5′ end of thepilin gene and the 3′ end of the neighboring gene (Nicotinate-nucleotidepyrophosphorylase) in the Pseudomonas genome (to be described in greaterdetail elsewhere). Results revealed the following: most strainsexhibited a 12 amino acid loop while one, SBI-N, had a 17 amino acidloop. Strains 82932 and 82935 had the same loop sequence as KB7(accession No, Q53391) and 90063 had a loop that matched PAO1 (accessionNo, A25023). Strains 1071 and SBI-N exhibited loops with novel sequences(See Tables 1 and 2). Strain M2, a mouse isolate, was not sequenced.

[0268] B. Toxin Neutralizing Response

[0269] The inhibition of protein synthesis of purified PE64 and PE64pilon eukaryotic cells in culture was determined as described in Ogata etal., J Biol. Chem. 265(33):20678-85 (1990). For inactivating cytotoxicactivity, the PE64pil proteins were incubated 30 min at 22° C. withrabbit sera, containing anti PE64Δ553pil antibodies, prior they wereadded to L929 or A549 cells in 24 well tissue culture dishes.

[0270] Rabbit antisera were evaluated for toxin neutralizing activity.All four of the immunized rabbits at a 1:20 dilution of sera neutralized1.0 μg/ml of toxin completely (FIG. 8). From these results, it wasconcluded that PE-pilin vaccine can generate antibodies of tworeactivities: one that blocks adhesion and one that neutralizes theexotoxin.

[0271] The present invention provides novel materials and methods forchimeric proteins comprising a non-toxic Pseudomonas exotoxin A and aType IV pilin loop sequence. While specific examples have been provided,the above description is illustrative and not restrictive. Any one ormore of the features of the previously described embodiments can becombined in any manner with one or more features of any otherembodiments in the present invention. Furthermore, many variations ofthe invention will become apparent to those skilled in the art uponreview of the specification. The scope of the invention should,therefore, be determined not with reference to the above description,but instead should be determined with reference to the appended claimsalong with their full scope of equivalents.

[0272] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

1 36 1 1839 DNA Pseudomonas aeruginosa CDS (1)..(1839) mature form ofExotoxin A 1 gcc gaa gaa gct ttc gac ctc tgg aac gaa tgc gcc aaa gcc tgcgtg 48 Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys Ala Cys Val 15 10 15 ctc gac ctc aag gac ggc gtg cgt tcc agc cgc atg agc gtc gac ccg96 Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser Val Asp Pro 20 2530 gcc atc gcc gac acc aac ggc cag ggc gtg ctg cac tac tcc atg gtc 144Ala Ile Ala Asp Thr Asn Gly Gln Gly Val Leu His Tyr Ser Met Val 35 40 45ctg gag ggc ggc aac gac gcg ctc aag ctg gcc atc gac aac gcc ctc 192 LeuGlu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp Asn Ala Leu 50 55 60 agcatc acc agc gac ggc ctg acc atc cgc ctc gaa ggc ggc gtc gag 240 Ser IleThr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly Gly Val Glu 65 70 75 80 ccgaac aag ccg gtg cgc tac agc tac acg cgc cag gcg cgc ggc agt 288 Pro AsnLys Pro Val Arg Tyr Ser Tyr Thr Arg Gln Ala Arg Gly Ser 85 90 95 tgg tcgctg aac tgg ctg gta ccg atc ggc cac gag aag ccc tcg aac 336 Trp Ser LeuAsn Trp Leu Val Pro Ile Gly His Glu Lys Pro Ser Asn 100 105 110 atc aaggtg ttc atc cac gaa ctg aac gcc ggc aac cag ctc agc cac 384 Ile Lys ValPhe Ile His Glu Leu Asn Ala Gly Asn Gln Leu Ser His 115 120 125 atg tcgccg atc tac acc atc gag atg ggc gac gag ttg ctg gcg aag 432 Met Ser ProIle Tyr Thr Ile Glu Met Gly Asp Glu Leu Leu Ala Lys 130 135 140 ctg gcgcgc gat gcc acc ttc ttc gtc agg gcg cac gag agc aac gag 480 Leu Ala ArgAsp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn Glu 145 150 155 160 atgcag ccg acg ctc gcc atc agc cat gcc ggg gtc agc gtg gtc atg 528 Met GlnPro Thr Leu Ala Ile Ser His Ala Gly Val Ser Val Val Met 165 170 175 gcccag acc cag ccg cgc cgg gaa aag cgc tgg agc gaa tgg gcc agc 576 Ala GlnThr Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190 ggcaag gtg ttg tgc ctg ctc gac ccg ctg gac ggg gtc tac aac tac 624 Gly LysVal Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr 195 200 205 ctcgcc cag caa cgc tgc aac ctc gac gat acc tgg gaa ggc aag atc 672 Leu AlaGln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile 210 215 220 taccgg gtg ctc gcc ggc aac ccg gcg aag cat gac ctg gac atc aaa 720 Tyr ArgVal Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys 225 230 235 240ccc acg gtc atc agt cat cgc ctg cac ttt ccc gag ggc ggc agc ctg 768 ProThr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu 245 250 255gcc gcg ctg acc gcg cac cag gct tgc cac ctg ccg ctg gag act ttc 816 AlaAla Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe 260 265 270acc cgt cat cgc cag ccg cgc ggc tgg gaa caa ctg gag cag tgc ggc 864 ThrArg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly 275 280 285tat ccg gtg cag cgg ctg gtc gcc ctc tac ctg gcg gcg cgg ctg tcg 912 TyrPro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser 290 295 300tgg aac cag gtc gac cag gtg atc cgc aac gcc ctg gcc agc ccc ggc 960 TrpAsn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly 305 310 315320 agc ggc ggc gac ctg ggc gaa gcg atc cgc gag cag ccg gag cag gcc 1008Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala 325 330335 cgt ctg gcc ctg acc ctg gcc gcc gcc gag agc gag cgc ttc gtc cgg 1056Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg 340 345350 cag ggc acc ggc aac gac gag gcc ggc gcg gcc aac gcc gac gtg gtg 1104Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val 355 360365 agc ctg acc tgc ccg gtc gcc gcc ggt gaa tgc gcg ggc ccg gcg gac 1152Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp 370 375380 agc ggc gac gcc ctg ctg gag cgc aac tat ccc act ggc gcg gag ttc 1200Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe 385 390395 400 ctc ggc gac ggc ggc gac gtc agc ttc agc acc cgc ggc acg cag aac1248 Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn 405410 415 tgg acg gtg gag cgg ctg ctc cag gcg cac cgc caa ctg gag gag cgc1296 Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg 420425 430 ggc tat gtg ttc gtc ggc tac cac ggc acc ttc ctc gaa gcg gcg caa1344 Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln 435440 445 agc atc gtc ttc ggc ggg gtg cgc gcg cgc agc cag gac ctc gac gcg1392 Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala 450455 460 atc tgg cgc ggt ttc tat atc gcc ggc gat ccg gcg ctg gcc tac ggc1440 Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly 465470 475 480 tac gcc cag gac cag gaa ccc gac gca cgc ggc cgg atc cgc aacggt 1488 Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly485 490 495 gcc ctg ctg cgg gtc tat gtg ccg cgc tcg agc ctg ccg ggc ttctac 1536 Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr500 505 510 cgc acc agc ctg acc ctg gcc gcg ccg gag gcg gcg ggc gag gtcgaa 1584 Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu515 520 525 cgg ctg atc ggc cat ccg ctg ccg ctg cgc ctg gac gcc atc accggc 1632 Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly530 535 540 ccc gag gag gaa ggc ggg cgc ctg gag acc att ctc ggc tgg ccgctg 1680 Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu545 550 555 560 gcc gag cgc acc gtg gtg att ccc tcg gcg atc ccc acc gacccg cgc 1728 Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp ProArg 565 570 575 aac gtc ggc ggc gac ctc gac ccg tcc agc atc ccc gac aaggaa cag 1776 Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys GluGln 580 585 590 gcg atc agc gcc ctg ccg gac tac gcc agc cag ccc ggc aaaccg ccg 1824 Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys ProPro 595 600 605 cgc gag gac ctg aag 1839 Arg Glu Asp Leu Lys 610 2 613PRT Pseudomonas aeruginosa 2 Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu CysAla Lys Ala Cys Val 1 5 10 15 Leu Asp Leu Lys Asp Gly Val Arg Ser SerArg Met Ser Val Asp Pro 20 25 30 Ala Ile Ala Asp Thr Asn Gly Gln Gly ValLeu His Tyr Ser Met Val 35 40 45 Leu Glu Gly Gly Asn Asp Ala Leu Lys LeuAla Ile Asp Asn Ala Leu 50 55 60 Ser Ile Thr Ser Asp Gly Leu Thr Ile ArgLeu Glu Gly Gly Val Glu 65 70 75 80 Pro Asn Lys Pro Val Arg Tyr Ser TyrThr Arg Gln Ala Arg Gly Ser 85 90 95 Trp Ser Leu Asn Trp Leu Val Pro IleGly His Glu Lys Pro Ser Asn 100 105 110 Ile Lys Val Phe Ile His Glu LeuAsn Ala Gly Asn Gln Leu Ser His 115 120 125 Met Ser Pro Ile Tyr Thr IleGlu Met Gly Asp Glu Leu Leu Ala Lys 130 135 140 Leu Ala Arg Asp Ala ThrPhe Phe Val Arg Ala His Glu Ser Asn Glu 145 150 155 160 Met Gln Pro ThrLeu Ala Ile Ser His Ala Gly Val Ser Val Val Met 165 170 175 Ala Gln ThrGln Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 180 185 190 Gly LysVal Leu Cys Leu Leu Asp Pro Leu Asp Gly Val Tyr Asn Tyr 195 200 205 LeuAla Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu Gly Lys Ile 210 215 220Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu Asp Ile Lys 225 230235 240 Pro Thr Val Ile Ser His Arg Leu His Phe Pro Glu Gly Gly Ser Leu245 250 255 Ala Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu ThrPhe 260 265 270 Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu GlnCys Gly 275 280 285 Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala AlaArg Leu Ser 290 295 300 Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala LeuAla Ser Pro Gly 305 310 315 320 Ser Gly Gly Asp Leu Gly Glu Ala Ile ArgGlu Gln Pro Glu Gln Ala 325 330 335 Arg Leu Ala Leu Thr Leu Ala Ala AlaGlu Ser Glu Arg Phe Val Arg 340 345 350 Gln Gly Thr Gly Asn Asp Glu AlaGly Ala Ala Asn Ala Asp Val Val 355 360 365 Ser Leu Thr Cys Pro Val AlaAla Gly Glu Cys Ala Gly Pro Ala Asp 370 375 380 Ser Gly Asp Ala Leu LeuGlu Arg Asn Tyr Pro Thr Gly Ala Glu Phe 385 390 395 400 Leu Gly Asp GlyGly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn 405 410 415 Trp Thr ValGlu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg 420 425 430 Gly TyrVal Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln 435 440 445 SerIle Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala 450 455 460Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly 465 470475 480 Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly485 490 495 Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly PheTyr 500 505 510 Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly GluVal Glu 515 520 525 Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp AlaIle Thr Gly 530 535 540 Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile LeuGly Trp Pro Leu 545 550 555 560 Ala Glu Arg Thr Val Val Ile Pro Ser AlaIle Pro Thr Asp Pro Arg 565 570 575 Asn Val Gly Gly Asp Leu Asp Pro SerSer Ile Pro Asp Lys Glu Gln 580 585 590 Ala Ile Ser Ala Leu Pro Asp TyrAla Ser Gln Pro Gly Lys Pro Pro 595 600 605 Arg Glu Asp Leu Lys 610 3 14PRT Artificial Sequence Description of Artificial SequencePseudomonasaeruginosa (PAK strain) Type IV short pilin loop 3 Cys Thr Ser Asp GlnAsp Glu Gln Phe Ile Pro Lys Gly Cys 1 5 10 4 14 PRT Artificial SequenceDescription of Artificial SequencePseudomonas aeruginosa (T2A strain)Type IV short pilin loop 4 Cys Thr Ser Thr Gln Asp Glu Met Phe Ile ProLys Gly Cys 1 5 10 5 14 PRT Artificial Sequence Description ofArtificial SequencePseudomonas aeruginosa (PAO and 90063 strains) TypeIV short pilin loop 5 Cys Lys Ser Thr Gln Asp Pro Met Phe Thr Pro LysGly Cys 1 5 10 6 14 PRT Artificial Sequence Description of ArtificialSequencePseudomonas aeruginosa (CD and PA103 strains) Type IV shortpilin loop 6 Cys Thr Ser Thr Gln Glu Glu Met Phe Ile Pro Lys Gly Cys 1 510 7 14 PRT Artificial Sequence Description of ArtificialSequencePseudomonas aeruginosa (K122-4 strain) Type IV short pilin loop7 Cys Thr Ser Asn Ala Asp Asn Lys Tyr Leu Pro Lys Thr Cys 1 5 10 8 14PRT Artificial Sequence Description of Artificial SequencePseudomonasaeruginosa (KB7, 82932 and 82935 strains) Type IV short pilin loop 8 CysAla Thr Thr Val Asp Ala Lys Phe Arg Pro Asn Gly Cys 1 5 10 9 14 PRTArtificial Sequence Description of Artificial SequencePseudomonasaeruginosa (1071 strain) Type IV short pilin loop 9 Cys Glu Ser Thr GlnAsp Pro Met Phe Thr Pro Lys Gly Cys 1 5 10 10 19 PRT Artificial SequenceDescription of Artificial SequencePseudomonas aeruginosa (577B strain)Type IV long pilin loop 10 Cys Asn Ile Thr Lys Thr Pro Thr Ala Trp LysPro Asn Tyr Ala Pro 1 5 10 15 Ala Asn Cys 11 19 PRT Artificial SequenceDescription of Artificial SequencePseudomonas aeruginosa (1244, 9D2 andP1 strains) Type IV long pilin loop 11 Cys Lys Ile Thr Lys Thr Pro ThrAla Trp Lys Pro Asn Tyr Ala Pro 1 5 10 15 Ala Asn Cys 12 19 PRTArtificial Sequence Description of Artificial SequencePseudomonasaeruginosa (SBI-N strain) Type IV long pilin loop 12 Cys Gly Ile Thr GlySer Pro Thr Asn Trp Lys Ala Asn Tyr Ala Pro 1 5 10 15 Ala Asn Cys 13 33PRT Artificial Sequence Description of Artificial SequenceNeisseriameningitidis (Z49820 strain) Type IV pilin loop 13 Cys Gly Leu Pro ValAla Arg Asp Asp Thr Asp Ser Ala Thr Asp Val 1 5 10 15 Lys Ala Asp ThrThr Asp Asn Ile Asn Thr Lys His Leu Pro Ser Thr 20 25 30 Cys 14 37 PRTArtificial Sequence Description of Artificial SequenceNeisseriameningitidis (Z69262 strain) Type IV pilin loop 14 Cys Gly Gln Pro ValThr Arg Gly Ala Gly Asn Ala Gly Lys Ala Asp 1 5 10 15 Asp Val Thr LysAla Gly Asn Asp Asn Glu Lys Ile Asn Thr Lys His 20 25 30 Leu Pro Ser ThrCys 35 15 31 PRT Artificial Sequence Description of ArtificialSequenceNeisseria meningitidis (Z69261 strain) Type IV pilin loop 15 CysGly Gln Pro Val Thr Arg Ala Lys Ala Asp Ala Asp Ala Ala Gly 1 5 10 15Lys Asp Thr Thr Asn Ile Asp Thr Lys His Leu Pro Ser Thr Cys 20 25 30 1633 PRT Artificial Sequence Description of Artificial SequenceNeisseriagonorrhoeae (pilE; X66144 strain) Type IV pilin loop 16 Cys Gly Gln ProVal Thr Arg Thr Gly Asp Asn Asp Asp Thr Val Ala 1 5 10 15 Asp Ala LysAsp Gly Lys Glu Ile Asp Thr Lys His Leu Pro Ser Thr 20 25 30 Cys 17 35PRT Artificial Sequence Description of Artificial SequenceNeisseriagonorrhoeae (pilE; AF043648 strain) Type IV pilin loop 17 Cys Gly GlnPro Val Lys Arg Asp Ala Gly Ala Lys Thr Gly Ala Asp 1 5 10 15 Asp ValLys Ala Asp Gly Asn Asn Gly Ile Asn Thr Lys His Leu Pro 20 25 30 Ser ThrCys 35 18 67 PRT Artificial Sequence Description of ArtificialSequenceVibrio cholera (U09807 strain) Type IV pilin loop 18 Cys Lys ThrLeu Val Thr Ser Val Gly Asp Met Phe Pro Phe Ile Asn 1 5 10 15 Val LysGlu Gly Ala Phe Ala Ala Val Ala Asp Leu Gly Asp Phe Glu 20 25 30 Thr SerVal Ala Asp Ala Ala Thr Gly Ala Gly Val Ile Lys Ser Ile 35 40 45 Ala ProGly Ser Ala Asn Leu Asn Leu Thr Asn Ile Thr His Val Glu 50 55 60 Lys LeuCys 65 19 67 PRT Artificial Sequence Description of ArtificialSequenceVibrio cholera (X64098 strain) Type IV pilin loop 19 Cys Lys ThrLeu Ile Thr Ser Val Gly Asp Met Phe Pro Tyr Ile Ala 1 5 10 15 Ile LysAla Gly Gly Ala Val Ala Leu Ala Asp Leu Gly Asp Phe Glu 20 25 30 Asn SerAla Ala Ala Ala Glu Thr Gly Val Gly Val Ile Lys Ser Ile 35 40 45 Ala ProAla Ser Lys Asn Leu Asp Leu Thr Asn Ile Thr His Val Glu 50 55 60 Lys LeuCys 65 20 13 PRT Artificial Sequence Description of ArtificialSequencePasteurella multocida (AF154834 strain) Type IV pilin loop 20Cys Asn Gly Gly Ser Glu Val Phe Pro Ala Gly Phe Cys 1 5 10 21 5 PRTArtificial Sequence Description of Artificial Sequenceendoplasmicreticulum (ER) retention domain in native Pseudomonas exotoxin A 21 ArgGlu Asp Leu Lys 1 5 22 4 PRT Artificial Sequence Description ofArtificial Sequenceendoplasmic reticulum (ER) retention domain 22 ArgGlu Asp Leu 1 23 4 PRT Artificial Sequence Description of ArtificialSequenceendoplasmic reticulum (ER) retention domain 23 Arg Glu Asp Leu 124 25 DNA Artificial Sequence Description of Artificial SequenceForwardprimer 24 ggcccatatg cacctgatac cccat 25 25 24 DNA Artificial SequenceDescription of Artificial SequenceReverse primer 25 gaattcagttacttcaggtc ctcg 24 26 28 DNA Artificial Sequence Description ofArtificial SequenceForward primer 26 ggcccatatg gagggcggca gcctggcc 2827 24 DNA Artificial Sequence Description of Artificial SequenceReverseprimer 27 gaattcagtt acttcaggtc ctcg 24 28 26 DNA Artificial SequenceDescription of Artificial SequencePCR primer pilATG (26 nc) 28gagatattca tgaaagctca aaaagg 26 29 20 DNA Artificial SequenceDescription of Artificial SequencePCR primer nadB4 (20 nc) 29 atctccatcggcaccctgac 20 30 21 DNA Artificial Sequence Description of ArtificialSequencePCR primer nadB1 (21 nc) 30 tggaagtgga agtggagaac c 21 31 90 DNAArtificial Sequence Description of Artificial Sequencecoding strand ofduplex 31 tggccctgac cctggccgcc gccgagagcg agcgcttcgt ccggcagggcaccggcaacg 60 acgaggccgg cgcggcaaac ctgcagggcc 90 32 54 DNA ArtificialSequence Description of Artificial SequenceSense oligonucleotide 32ttgtactagt gatcaggatg aacagtttat tccgaaaggt tgttcacgta tgca 54 33 54 DNAArtificial Sequence Description of Artificial SequenceAntisenseoligonucleotide 33 tacgtgaaca acctttcgga ataaactgtt catcctgatcactagtacaa tgca 54 34 17 PRT Artificial Sequence Description ofArtificial Sequencelong C-terminal peptide, peptide 1, amino acids128-142 of PAK strain 34 Xaa Cys Thr Ser Asp Gln Asp Glu Gln Phe Ile ProLys Gly Cys Ser 1 5 10 15 Xaa 35 7 PRT Artificial Sequence Descriptionof Artificial Sequencecore peptide, peptide 2, amino acids 134-140 ofPAK strain 35 Xaa Glu Gln Phe Ile Pro Xaa 1 5 36 7 PRT ArtificialSequence Description of Artificial Sequencescrambled peptide 36 Xaa IleAsp Pro Glu Phe Xaa 1 5

What is claimed is:
 1. A chimeric protein comprising: a non-toxicPseudomonas exotoxin A sequence and a Type IV pilin loop sequence, theType IV pilin loop sequence being located within the non-toxicPseudomonas exotoxin A sequence, wherein the chimeric protein is capableof reducing adherence of a microorganism expressing the Type IV pilinloop sequence to epithelial cells, and further wherein the chimericprotein, when introduced into a host, is capable of generatingpolyclonal antisera that reduce adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells. 2.The chimeric protein of claim 1, wherein the chimeric protein, whenintroduced into the host, is also capable of generating polyclonalantisera that neutralize cytotoxicity of Pseudomonas exotoxin A.
 3. Thechimeric protein of claim 1, wherein the non-toxic Pseudomonas exotoxinA sequence comprises: (a) a translocation domain sufficient to effecttranslocation of the chimeric protein to a cell cytosol; and (b) anendoplasmic reticulum retention domain that functions to translocate thechimeric protein from endosome to endoplasmic reticulum.
 4. The chimericprotein of claim 3, wherein the chimeric protein further comprises acell recognition domain that functions as a ligand for a cell surfacereceptor and that mediates binding of the chimeric protein to a cell. 5.The chimeric protein of claim 4, wherein the Type IV pilin loop sequenceis located between the translocation domain and the endoplasmicreticulum retention domain.
 6. The chimeric protein of claim 5, whereinthe Type IV pilin loop sequence comprises cysteine residues at both theN- and C-termini of the Type IV pilin loop sequence.
 7. The chimericprotein of claim 5, wherein the Type IV pilin loop sequence is frombacteria or yeast.
 8. The chimeric protein of claim 7, wherein the TypeIV pilin loop sequence is from Pseudomonas aeruginosa, Neisseriameningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurellamultocidam, or Candida.
 9. The chimeric protein of claim 8, wherein theType IV pilin loop sequence is from Pseudomonas aeruginosa.
 10. Thechimeric protein of claim 9, wherein the Type IV pilin loop sequence isselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:10.
 11. The chimeric protein of claim 5, wherein the translocationdomain comprises amino acids 280 to 364 of domain II of Pseudomonasexotoxin A.
 12. The chimeric protein of claim 5, wherein thetranslocation domain is domain II of Pseudomonas exotoxin A.
 13. Thechimeric protein of claim 5, wherein the endoplasmic reticulum retentiondomain is domain III of Pseudomonas exotoxin A except that amino acidGlu at position of 553 is deleted.
 14. The chimeric protein of claim 1,wherein the chimeric protein comprises more than one Type IV pilin loopsequence.
 15. The chimeric protein of claim 5, wherein the cellrecognition domain is domain Ia of Pseudomonas exotoxin A.
 16. Thechimeric protein of claim 5, wherein the cell recognition domain bindsto α2-macroglobulin receptor, epidermal growth factor receptor,transferrin receptor, interleukin-2 receptor, interleukin-6 receptor,interleukin-8 receptor, Fc receptor, poly-IgG receptor,asialoglycoprotein receptor, CD3, CD4, CD8, chemokine receptor, CD25,CD11B, CD11C, CD80, CD86, TNFalpha receptor, TOLL receptor, M-CSFreceptor, GM-CSF receptor, scavenger receptor, VEGF receptor, orcytokine receptor.
 17. A chimeric protein comprising: (a) a non-toxicPseudomonas exotoxin A sequence comprising domain Ia, domain II, anddomain III; and (b) a Type IV pilin loop sequence, wherein the Type IVpilin loop sequence is located between domain II and domain III of thenon-toxic Pseudomonas exotoxin A sequence.
 18. The chimeric protein ofclaim 17, wherein the non-toxic Pseudomonas exotoxin A sequence has theamino acid sequence of SEQ ID NO:2 with ΔE553.
 19. The chimeric proteinof claim 17, wherein the Type IV pilin loop sequence is from Pseudomonasaeruginosa, Neisseria meningtidis, Neisseria gonorrhoeae, Vibro cholera,Pasteurella multocidam, or Candida.
 20. The chimeric protein of claim17, wherein the Type IV pilin loop sequence is from Pseudomonasaeruginosa.
 21. A polynucleotide encoding a chimeric protein, thechimeric protein comprising: a non-toxic Pseudomonas exotoxin A sequenceand a Type IV pilin loop sequence, the Type IV pilin loop sequence beinglocated within the non-toxic Pseudomonas exotoxin A sequence, whereinthe chimeric protein is capable of reducing adherence of a microorganismexpressing the Type IV pilin loop sequence to epithelial cells, andfurther wherein the chimeric protein, when introduced into a host, iscapable of generating polyclonal antisera that prevent adherence of themicroorganism expressing the Type IV pilin loop sequence to theepithelial cells.
 22. The polynucleotide of claim 21, wherein thechimeric protein, when introduced into the host, is also capable ofgenerating polyclonal antisera that neutralize cytotoxicity ofPseudomonas exotoxin A.
 23. The polynucleotide of claim 21, wherein thenon-toxic Pseudomonas exotoxin A sequence comprises: (a) a translocationdomain sufficient to effect translocation of the chimeric protein to acell cytosol; and (b) an endoplasmic reticulum retention domain thatfunctions to translocate the chimeric protein from endosome toendoplasmic reticulum.
 24. The polynucleotide of claim 23, wherein thechimeric protein further comprises a cell recognition domain thatfunctions as a ligand for a cell surface receptor and that mediatesbinding of the chimeric protein to a cell.
 25. The polynucleotide ofclaim 24, wherein the Type IV pilin loop sequence is located between thetranslocation domain and the endoplasmic reticulum retention domain. 26.The polynucleotide of claim 25, wherein the Type IV pilin loop sequencecomprises cysteine residues at both the N- and C-termini of the Type IVpilin loop sequence.
 27. The polynucleotide of claim 25, wherein theType IV pilin loop sequence is from bacteria or yeast.
 28. Thepolynucleotide of claim 27, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa, Neisseria meningtidis, Neisseriagonorrhoeae, Vibro cholera, Pasteurella multocidam, or Candida.
 29. Thepolynucleotide of claim 28, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa.
 30. The polynucleotide of claim 29, whereinthe Type IV pilin loop sequence is selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10.
 31. The polynucleotide of claim 25,wherein the translocation domain comprises amino acids 280 to 364 ofdomain II of Pseudomonas exotoxin A.
 32. The polynucleotide of claim 25,wherein the translocation domain is domain II of Pseudomonas exotoxin A.33. The polynucleotide of claim 25, wherein the endoplasmic reticulumretention domain is domain III of Pseudomonas exotoxin A except thatamino acid Glu at position of 553 is deleted.
 34. The polynucleotide ofclaim 25, wherein the cell recognition domain is domain Ia ofPseudomonas exotoxin A.
 35. The polynucleotide of claim 25, wherein thecell recognition domain binds to α2-macroglobulin receptor, epidermalgrowth factor receptor, transferring receptor, Fc receptor, poly-IgGreceptor, asialoglycoprotein receptor, CD3, CD4, CD8, chemokinereceptor, CD25, CD11B, CD11C, CD80, CD86, TNFalpha receptor, TOLLreceptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, VEGFreceptor, or cytokine receptor.
 36. A polynucleotide encoding a chimericprotein, the chimeric protein comprising: (a) a non-toxic Pseudomonasexotoxin A sequence comprising domain Ia, domain II, and domain III; and(b) a Type IV pilin loop sequence, wherein the Type IV pilin loopsequence is located between domain II and domain III of the non-toxicPseudomonas exotoxin A sequence.
 37. The polynucleotide of claim 36,wherein the non-toxic Pseudomonas exotoxin A sequence has the amino acidsequence of SEQ ID NO:2 with ΔE553.
 38. The polynucleotide of claim 36,wherein the Type IV pilin loop sequence is from Pseudomonas aeruginosa,Neisseria meningtidis, Neisseria gonorrhoeae, Vibro cholera, Pasteurellamultocidam, or Candida.
 39. The polynucleotide of claim 36, wherein theType IV pilin loop sequence is from Pseudomonas aeruginosa.
 40. Anexpression cassette comprising the polynucleotide of claim
 21. 41. Acell comprising the expression cassette of claim
 40. 42. A compositioncomprising a chimeric protein, the chimeric protein comprising: anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A sequence, wherein the chimeric proteinis capable of reducing adherence of a microorganism expressing the TypeIV pilin loop sequence to epithelial cells, and further wherein thechimeric protein, when introduced into a host, is capable of generatingpolyclonal antisera that prevent adherence of the microorganismexpressing the Type IV pilin loop sequence to the epithelial cells. 43.The composition of claim 42, wherein the chimeric protein, whenintroduced into the host, is also capable of generating polyclonalantisera that neutralize cytotoxicity of Pseudomonas exotoxin A.
 44. Thecomposition of claim 42, wherein the composition further comprises apharmacologically acceptable carrier.
 45. The composition of claim 42,wherein the composition is formulated as a nasal or oral spray.
 46. Thecomposition of claim 42, wherein the non-toxic Pseudomonas exotoxin Asequence comprises: (a) a translocation domain sufficient to effecttranslocation of the chimeric protein to a cell cytosol; and (b) anendoplasmic reticulum retention domain that functions to translocate thechimeric protein from endosome to endoplasmic reticulum.
 47. Thecomposition of claim 46, wherein the chimeric protein further comprisesa cell recognition domain that functions as a ligand for a cell surfacereceptor and that mediates binding of the chimeric protein to a cell.48. The composition of claim 47, wherein the Type IV pilin loop sequenceis from Pseudomonas aeruginosa.
 49. A method for eliciting an immuneresponse in a host, the method comprising the step of administering tothe host an immunologically effective amount of a composition comprisinga chimeric protein comprising: a non-toxic Pseudomonas exotoxin Asequence and a Type IV pilin loop sequence, the Type IV pilin loopsequence being located within the non-toxic Pseudomonas exotoxin Asequence, wherein the chimeric protein is capable of reducing adherenceof a microorganism expressing the Type IV pilin loop sequence toepithelial cells, and further wherein the chimeric protein, whenintroduced into the host, is capable of generating polyclonal antiserathat prevent adherence of the microorganism expressing the Type IV pilinloop sequence to the epithelial cells.
 50. The method of claim 49,wherein the chimeric protein, when introduced into the host, is capableof generating polyclonal antisera that neutralize cytotoxicity ofPseudomonas exotoxin A.
 51. The method of claim 49, wherein the host isa human.
 52. The method of claim 49, wherein the non-toxic Pseudomonasexotoxin A sequence comprises: (a) a translocation domain sufficient toeffect translocation of the chimeric protein to a cell cytosol; and (b)an endoplasmic reticulum retention domain that functions to translocatethe chimeric protein from endosome to endoplasmic reticulum.
 53. Themethod of claim 52, wherein the chimeric protein further comprises acell recognition domain that functions as a ligand for a cell surfacereceptor and that mediates binding of the chimeric protein to a cell.54. The method of claim 53, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa.
 55. A method of eliciting an immuneresponse in a host, the method comprising the step of administering tothe host an immunologically effective amount of an expression cassettecomprising a polynucleotide encoding a chimeric protein comprising: anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A, wherein the chimeric protein iscapable of reducing adherence of a microorganism expressing the Type IVpilin loop sequence to epithelial cells, and further wherein thechimeric protein, when introduced into the host, is capable ofgenerating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to theepithelial cells.
 56. The method of claim 55, wherein the chimericprotein, when introduced into the host, is capable of generatingpolyclonal antisera that neutralize cytotoxicity of Pseudomonas exotoxinA.
 57. The method of claim 55, wherein the host is a human.
 58. Themethod of claim 55, wherein the non-toxic Pseudomonas exotoxin Asequence comprises: (a) a translocation domain sufficient to effecttranslocation of the chimeric protein to a cell cytosol; and (b) anendoplasmic reticulum retention domain that functions to translocate thechimeric protein from endosome to endoplasmic reticulum.
 59. The methodof claim 58, wherein the chimeric protein further comprises a cellrecognition domain that functions as a ligand for a cell surfacereceptor and that mediates binding of the chimeric protein to a cell.60. The method of claim 59, wherein the Type IV pilin loop sequence isfrom Pseudomonas aeruginosa.
 61. A method of generating antibodiesspecific for a Type IV pilin loop sequence, comprising introducing intoa host a composition comprising a chimeric protein comprising anon-toxic Pseudomonas exotoxin A sequence and a Type IV pilin loopsequence, the Type IV pilin loop sequence being located within thenon-toxic Pseudomonas exotoxin A, wherein the chimeric protein iscapable of reducing adherence of a microorganism expressing the Type IVpilin loop sequence to epithelial cells, and further wherein thechimeric protein, when introduced into the host, is capable ofgenerating polyclonal antisera that reduce adherence of themicroorganism expressing the Type IV pilin loop sequence to epithelialcells.
 62. The method of claim 61, wherein the chimeric protein, whenintroduced into the host, is capable of generating polyclonal antiserathat neutralize cytotoxicity of Pseudomonas exotoxin A.
 63. The methodof claim 61, wherein the host is a human.
 64. The method of claim 61,wherein the non-toxic Pseudomonas exotoxin A sequence comprises: (a) atranslocation domain sufficient to effect translocation of the chimericprotein to a cell cytosol; and (b) an endoplasmic reticulum retentiondomain that functions to translocate the chimeric protein from endosometo endoplasmic reticulum.
 65. The method of claim 64, wherein thechimeric protein further comprises a cell recognition domain thatfunctions as a ligand for a cell surface receptor and that mediatesbinding of the chimeric protein to a cell.
 66. The method of claim 65,wherein the Type IV pilin loop sequence is from Pseudomonas aeruginosa.